Viral encoded semaphorin protein receptor DNA and polypeptides

ABSTRACT

The invention is directed to VESPR polypeptides as a purified and isolated protein, the DNA encoding the VESPR polypeptide, host cells transfected with cDNAs encoding VESPR, and methods for preparing VESPR polypeptides.

CROSS REFERENCE

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/459,061, filed Dec. 10, 1999, currently pending, which is adivisional of U.S. patent application Ser. No. 09/181,706, filed Oct.28, 1998, now issued as U.S. Pat. No. 6,130,068, which is a continuationof U.S. patent application Ser. No. 08/958,598, filed Oct. 28, 1997,converted to a provisional application, U.S. Patent Application No.60/112,009, on Oct. 26, 1998, now abandoned, the disclosures of whichare incorporated by reference herein.

FIELD OF THE INVENTION

[0002] The present invention relates to semaphorin receptorpolypeptides, the nucleic acids encoding such semaphorin receptorpolypeptides, processes for producing recombinant semaphorin receptorpolypeptides, and pharmaceutical compositions containing suchpolypeptides.

BACKGROUND OF THE INVENTION

[0003] The semaphorin gene family includes a large number of moleculesthat encode related transmembrane and secreted glycoproteins known to beneurologic regulators. The semaphorins are generally well conserved intheir extracellular domains which are typically about 500 amino acids inlength. Semaphorin family proteins have been observed in neuronal andnormeuronal tissue and have been studied largely for their role inneuronal growth cone guidance. For example, the secreted semaphorinsknown as collapsin-1 and Drosophila semaphorin II are selectivelyinvolved in repulsive growth cone guidance during development. Flieshaving semaphorin II genes that are mutated so that their function isreduced exhibit abnormal behavior characteristics.

[0004] Another semaphorin gene has been identified in several strains ofpoxvirus. This semaphorin is found in vaccinia virus (Copenhagen strain)and is encoded in an open reading frame (ORF) known as A39R. The A39Rencoded protein has no transmembrane domain and no potential membranelinkage and is known to be a secreted protein. A variola virus ORF alsocontains sequences that share homology with the vaccinia virus ORF A39Rat the nucleotide level and the amino acid level. Another viralsemaphorin, AHV-sema, has been found in the Alcelaphine Herpesvirus(AHV).

[0005] Genes encoding mammalian (human, rat, and mouse) semaphorins havebeen identified, based upon their similarity to insect semaphorins.Functional studies of these semaphorins suggest that embryonic and adultneurons require a semaphorin to establish workable connections.Significantly, the fast response time of growth cone cultures toappropriate semaphorins suggests that semaphorin signaling involves areceptor-mediated signal transduction mechanism. To date, one semaphorinreceptor, designated neuropilin, has been isolated using mRNA from ratspinal cord. Another receptor, designated neuropilin-2, has beensuggested (Kolodkin et al. Cell 90:753-762, 1997)

[0006] Semaphorin ligands that are secreted into the extracellularmilieu signal through receptor bearing cells in a local and systemicfashion. In order to further investigate the nature of cellularprocesses regulated by such local and systemic signaling, it would bebeneficial to identify additional semaphorin receptors and ligands.Furthermore, because virus encoded semaphorins are produced by infectedcells and are present in viruses that are lytic (poxviruses) and virusesthat are not known to be neurotropic (AHV), it is unlikely that theirprimary function is to modify neurologic responses. It is more likelythat the virus encoded semaphorins function to modify the immunologicresponse of the infected host and it is likely that mammalian homologuesto virus encoded semaphorins function to modify the immunologicresponse. In view of the suggestion that viral semaphorins may functionin the immune system as natural immunoregulators it would be beneficialto identify semaphorin receptors as therapeutic agents for enhancing ordownregulating the immune response.

SUMMARY OF THE INVENTION

[0007] The present invention pertains to semaphorin receptors asisolated or homogeneous proteins. In particular, the present inventionprovides a semaphorin receptor polypeptide, designated VESPR (ViralEncoded Semaphorin Protein Receptor) that binds semaphorins, including,but not limited to, the A39R vaccinia semaphorin and AHV semaphorin.Also, within the scope of the present invention are DNAs encoding VESPRpolypeptides and expression vectors that include DNA encoding VESPRpolypeptides. The present invention also includes host cells that havebeen transfected or transformed with expression vectors that include DNAencoding a VESPR polypeptide, and processes for producing VESPRpolypeptides by culturing such host cells under conditions conducive toexpression. The present invention further includes antibodies directedagainst VESPR polypeptides.

[0008] Further within the scope of the present invention are processesfor purifying or separating semaphorins or cells that expresssemaphorins to which the VESPR polypeptides of the present inventionbind. Such processes include binding at least one VESPR polypeptide to asolid phase matrix and contacting a mixture containing a semaphorinpolypeptide to which the VESPR polypeptide binds, or a mixture of cellsexpressing the semaphorin with the bound VESPR polypeptide, and thenseparating the contacting surface and the solution.

[0009] The present invention additionally provides processes fortreating inflammation and inflammatory diseases. Such processes includeadministering a therapeutically effective amount of a soluble VESPRpolypeptide to an human or other mammal afflicted with a diseaseassociated with proinflammatory activity of a semaphorin ligand.

DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention provides novel semaphorin receptorpolypeptides designated Viral Encoded Semaphorin Protein Receptor(VESPR), DNA encoding VESPR polypeptides and recombinant expressionvectors that include DNA encoding VESPR polypeptides. The presentinvention additionally provides methods for isolating VESPR polypeptidesand methods for producing recombinant VESPR polypeptides by cultivatinghost cells transfected with the recombinant expression vectors underconditions appropriate for expressing semaphorin receptors andrecovering the expressed receptor polypeptide.

[0011] In particular, the present invention provides VESPR polypeptidesthat bind semaphorins, including but not limited to, the vaccinia virusA39R semaphorin and the AHV semaphorin. The native VESPR polypeptidedescribed herein was isolated using an Ectromelia virus A39Rsemaphorin/Fc fusion protein (A39R/Fc) to recover VESPR from themembranes of human cells expressing the receptor. As described in theexamples below, flow cytometry experiments establish that the VESPRpolypeptide polypeptides of the present invention are expressed by Bcells lines, monocyte-type cell lines, T cell lines, dendritic cells NKcells, lung epithelial cells, stroma, intestinal epithelial cells andlymphoma cells.

[0012] Furthermore, as demonstrated in the examples below, VESPRpolypeptides of the present invention bind with their ligands toparticipate in upregulating the CD69 activation antigen on dendriticcells. Also characteristic of semaphorin receptors described herein istheir ability to interact with their ligands to synergize withinterferon and SAC to upregulate IL-12 production and down regulate MHCclass II and CD86 expression on mouse dendritic cells. VESPRpolypeptides of the present invention are also associated with increasedexpression of CD54 on monocytes which suggests cellular activation as aresult of the interaction between semaphorins and their receptors. Amongthe uses of the VESPR polypeptides that flow from aforementionedbiological properties of the receptor-ligand interaction are inducingIL-12 production and subsequent natural killer cell activation. VESPRpolypeptide polypeptides find further use in treating diseases andadverse conditions associated with inflammation. In particular, solubleVESPR polypeptides can be used to antagonize proinflammatory activitiesassociated with the interaction of semaphorin ligands and theirreceptors. Rheumatoid arthritis, a disease associated with chronicinflammation of synovial tissue, has been linked with upregulation ofthe human semaphorin E gene (Mangasser-Stephan et al., Biochem andBiophys Res Com, 234:153-156, 1997). Thus, soluble forms of VESPRpolypeptides of the present invention may be useful in downregulatingsemaphorin activity that mediates this inflammatory disease.

[0013] VESPR, a native semaphorin receptor of the present invention wasisolated using a viral semaphorin ligand known as Ectromelia A39R.Example 1 below describes isolating the A39R semaphorin ligand andpreparing an A39R/Fc fusion protein which was used to identify celllines that bind the ligand and to determine the effects of interactionsbetween A39R and its cell bound receptor.

[0014] Examples 4 and 5 describe identifying a native VESPR polypeptideof the present invention and isolating and purifying a human VESPRpolypeptide. The amino acid sequence of the human VESPR polypeptide,isolated as described in Example 5, is disclosed in SEQ ID NO:2. Theamino acid sequence of SEQ ID NO: 2 was obtained by sequencing theisolated and purified receptor using tandem mass spectrometry analysisof peptides produced in a trypsin digestion, in combination withcontiguous EST sequences and identified cDNAs. The amino acid sequencepresented in SEQ ID NO:2 has a predicted extracellular domain of aminoacids 1-944 that includes a signal peptide with a cleavage sitepredicted at amino acid 34. The predicted transmembrane domain of SEQ IDNO:2 includes amino acids 945-965 and the cytoplasmic domain of SEQ IDNO:2 extends from amino acids 966-1568.

[0015] A DNA encoding amino acids 19-1100 of human VESPR in E. coliDH10B was deposited with the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209, U.S.A. on Oct. 22, 1997and assigned accession number 98560. The deposit was made under theterms of the Budapest Treaty. The DNA construct of the deposit differsfrom that of SEQ ID NO:1 in that nucleotide 172 is C. The resultingencoded amino acid 58 is leu.

[0016] Amino acid sequence searches were performed in available databases for proteins and polypeptides sharing homology with the fulllength VESPR or domains thereof. The searches for polypeptides sharinghomology with VESPR were performed using the BLAST algorithm describedby Altschul et al., J Mol Bio 215:403-410 (1990). This program was usedto compare the VESPR amino acid sequence with protein and DNA sequencesfound in data bases obtained from the National Center for BiotechnologyInformation. Similarity scores obtained as a result of these searchesidentified groups of polypeptides having varying degrees of homologywith VESPR. The highest degree of similarity was found to be between theVESPR and a group of proteins known as the “plexin gene family”(Maestrini et al., 1996, and Kameyama et al., 1996). Pairwise andmultiple sequence alignments between VESPR and human and murine membersof the plexin family were performed using the Smith-Waterman algorithmas implemented in the Genetics Computer Group (GCG) programs “BESTFIT”and “PILEUP” (Wisconsin Package, 9.0). The GCG program “DISTANCES” wasused to calculate average pairwise percentage identity of the alignedprotein sequences.

[0017] Pairwise sequence alignments between the VESPR polypeptide andeach of several members of the plexin gene family revealed an averageidentity in their cytoplasmic domain (amino acids 966-1568) of 39% to40% and an average identity for each of the entire protein of 24%-25%.The higher degree of homology in the cytoplasmic domains suggestssimilar signal transduction mechanisms among the cytoplasmic domains.

[0018] In order to identify regions of similarity between the proteinsequences found to have some overall homology, homology analyses of theresults of protein data base searches were performed using the BLIXEMand MSPCRUNCH programs (Sonnhammer and Durbin (1944a,b) The homologyanalyses revealed a novel subdomain with similarity to a region of thesemaphorin domain of a number of members of the semaphorin family ofgenes described by Kolodkin et al. (1993). The novel subdomain includesamino acids 380-482 of the VESPR sequence of the present invention. Thissubdomain can be subdivided into two distinct smaller regions, thatinclude residues 388-402 and 454-482, respectively. The C-proximalhalf-subdomain contains several highly conserved cysteine and tryptophanresidues, forming a consensus sequence ofC-x(5)-C-x(2)-C-x(7)-C-x-W-C-x(5)-C, where x is any amino acid. Thisentire subdomain is distinct from the canonical semaphorin domaindescribed for the semaphorin gene family in that (a) it is smaller (100amino acid residues for the subdomain vs 500 residues for the entiresemaphorin domain), (b) it is also present in the plexin gene family andMET-hepatocyte growth factor receptor family, neither of which is acanonical semaphorin gene family members, and (c). it is present in agene which is not itself a member of the semaphorin gene family butwhich interacts with a member of the semaphorin family (A39R). Thesesubdomain sequences, therefore, represent peptides that are potentiallycapable of further identifying other receptors which interact withsemaphorins.

[0019] A cDNA sequence that encodes the VESPR polypeptide of SEQ ID NO:2was assembled as a composite of contiguous EST and cloned cDNAnucleotide sequences and is disclosed in SEQ ID NO:1. As described inExample 5, identifying the cDNA that encodes the amino acid sequence ofSEQ ID NO:2 enables constructing expression vectors that include theencoding cDNAs. Then culturing host cells transfected with a recombinantexpression vector that contains cDNA encoding VESPR polypeptide, underconditions appropriate for expressing the VESPR polypeptide, andrecovering the expressed VESPR polypeptide provides methods forproducing VESPR polypeptides of the present invention.

[0020] Since VESPR polypeptide is found in B cell lines, T cell linesand dendritic cells, treating a variety of conditions associated withoveractive or underactive immuno-regulation is possible. Moreover, theligand and receptor complex may be involved in neural growth,development and/or maintenance. While not limited to such, particularuses of the VESPR are described infra.

[0021] The terms “VESPR” and “VESPR polypeptide” of the presentinvention encompass polypeptides having the amino acid sequence SEQ IDNO:2, and proteins that are encoded by nucleic acids that contain thenucleic acid sequence of SEQ ID NO:1. In addition, the terms includethose polypeptides that have a high degree of similarity or a highdegree of identity with the amino acid sequence of SEQ ID NO:2, whichpolypeptides are biologically active and bind at least one molecule orfragments of a molecule that are members of the semaphorin family. Inaddition, the term VESPR refers to biologically active gene products ofthe DNA of SEQ ID NO:1. Further encompassed by the term VESPR aresoluble or truncated proteins that comprise primarily the bindingportion of the protein, retain biological activity and are capable ofbeing secreted. Specific examples of such soluble proteins are thosecomprising the sequence of amino acids 1-944 of SEQ ID NO:2.

[0022] The term “biologically active” as it refers to VESPR orsemaphorin receptor polypeptide, means that the VESPR or semaphorinreceptor polypeptide is capable of binding to at least one semaphorin.Assays suitable for determining VESPR binding are described herein andcan include standard flow cytometry tests and slide binding tests.

[0023] “Isolated” means a VESPR is substantially free of associationwith other proteins or polypeptides residual of the expression process,for example, as a purification product of recombinant host cell cultureor as a purified extract.

[0024] A VESPR variant as referred to herein, means a polypeptidesubstantially homologous to native VESPR, but which has an amino acidsequence different from that of native VESPR because of one or moredeletions, insertions or substitutions. The variant amino acid sequencepreferably is at least 80% identical to a native VESPR amino acidsequence, most preferably at least 90% identical. The percent identitymay be determined, for example, by comparing sequence information usingthe GAP computer program, version 8.1 described by Devereux et al.(Nucl. Acids Res. 12:387, 1984) and available from the University ofWisconsin Genetics Computer Group (UWGCG). The preferred defaultparameters for the GAP program include: (1) a unary comparison matrix(containing a value of 1 for identities and 0 for non-identities) fornucleotides, and the weighted comparison matrix of Gribskov and Burgess,Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff,eds., Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for eachgap and an additional 0.10 penalty for each symbol in each gap; and (3)no penalty for end gaps. Variants may comprise conservativelysubstituted sequences, meaning that a given amino acid residue isreplaced by a residue having similar physiochemical characteristics.Examples of conservative substitutions include substitution of onealiphatic residue for another, such as Ile, Val, Leu, or Ala for oneanother, or substitutions of one polar residue for another, such asbetween Lys and Arg; Glu and Asp; or Gln and Asn. Other suchconservative substitutions, for example, substitutions of entire regionshaving similar hydrophobicity characteristics, are well known. Naturallyoccurring VESPR variants or alleles are also encompassed by theinvention. Examples of such variants are proteins that result fromalternate mRNA splicing events or from proteolytic cleavage of the VESPRprotein, wherein the binding property is retained. Alternate splicing ofmRNA may yield a truncated but biologically active VESPR polypeptide,such as a naturally occurring soluble form of the protein, for example.Variations attributable to proteolysis include, for example, differencesin the N-termini upon expression in different types of host cells, dueto proteolytic removal of one or more terminal amino acids from theVESPR polypeptide (generally from 1-5 terminal amino acids).

[0025] As mentioned above, Example 1 describes the construction of novelviral A39R/Fc fusion proteins useful in studying VESPR binding. Otherantibody Fc regions may be substituted for the human IgG1 Fc regiondescribed in the Example. Suitable Fc regions are those that can bindwith high affinity to protein A or protein G, and include the Fc regionof human IgG1 or fragments of the human or murine IgG1 Fc region, e.g.,fragments comprising at least the hinge region so that interchaindisulfide bonds will form. The viral A39R:Fc fusion protein offers theadvantage of being easily purified. In addition, disulfide bonds formbetween the Fc regions of two separate fusion protein chains, creatingdimers.

[0026] As described above, in one aspect, the present invention includessoluble VESPR polypeptides. Soluble VESPR polypeptides comprise all orpart of the extracellular domain of a native VESPR but lack thetransmembrane region that would cause retention of the polypeptide on acell membrane. Soluble VESPR polypeptides advantageously comprise thenative (or a heterologous) signal peptide when initially synthesized topromote secretion, but the signal peptide is cleaved upon secretion ofVESPR polypeptides from the cell. Soluble VESPR polypeptides encompassedby the invention retain the ability to bind semaphorin ligands. Indeed,soluble VESPR polypeptides may also include part of the signal or partof the cytoplasmic domain or other sequences, provided that the solubleVESPR protein can be secreted.

[0027] Soluble VESPR may be identified (and distinguished from itsnon-soluble membrane-bound counterparts) by separating intact cellswhich express the desired protein from the culture medium, e.g., bycentrifugation, and assaying the medium (supernatant) for the presenceof the desired protein. The presence of VESPR in the medium indicatesthat the protein was secreted from the cells and thus is a soluble formof the desired protein.

[0028] Soluble forms of VESPR polypeptides possess many advantages overthe native, membrane bound VESPR protein. Purification of the proteinsfrom recombinant host cells is feasible, since the soluble proteins aresecreted from the cells. Further, soluble proteins are generally moresuitable for intravenous administration.

[0029] Examples of soluble VESPR polypeptides include those comprising asubstantial portion of the extracellular domain of a native VESPRpolypeptide. An example of a soluble VESPR polypeptide is amino acids1-944 of SEQ ID NO:2. In addition, truncated soluble VESPR proteinscomprising less than the entire extracellular domain are included in theinvention, e.g. amino acids 35-944. When initially expressed within ahost cell, soluble VESPR polypeptides may additionally comprise one ofthe heterologous signal peptides described below that is functionalwithin the host cells employed. Alternatively, the protein may comprisethe native signal peptide. In one embodiment of the invention, solubleVESPR can be expressed as a fusion protein comprising (from N- toC-terminus) the yeast α-factor signal peptide, a FLAG® peptide describedbelow and in U.S. Pat. No. 5,011,912, and soluble VESPR polypeptideconsisting of amino acids 1-944 or 35-944 of SEQ ID NO:2. Thisrecombinant fusion protein is expressed in and secreted from yeastcells. The FLAG® peptide facilitates purification of the protein, andsubsequently may be cleaved from the soluble VESPR using bovine mucosalenterokinase. Isolated DNA sequences encoding soluble VESPR proteins areencompassed by the invention.

[0030] Truncated VESPR polypeptides, including soluble polypeptides, maybe prepared by any of a number of conventional techniques. A desired DNAsequence may be chemically synthesized using techniques known per se.DNA fragments also may be produced by restriction endonuclease digestionof a full length cloned DNA sequence, and isolated by electrophoresis onagarose gels. Linkers containing restriction endonuclease cleavagesite(s) may be employed to insert the desired DNA fragment into anexpression vector, or the fragment may be digested at cleavage sitesnaturally present therein. The well known polymerase chain reactionprocedure also may be employed to amplify a DNA sequence encoding adesired protein fragment. As a further alternative, known mutagenesistechniques may be employed to insert a stop codon at a desired point,e.g., immediately downstream of the codon for the last amino acid of thebinding domain.

[0031] As stated above, the invention provides isolated or homogeneousVESPR polypeptides, both recombinant and non-recombinant. Variants andderivatives of native VESPR proteins that retain the desired biologicalactivity (e.g., the ability to bind to semaphorins) may be obtained bymutations of nucleotide sequences coding for native VESPR polypeptides.Alterations of the native amino acid sequence may be accomplished by anyof a number of conventional methods. Mutations can be introduced atparticular loci by synthesizing oligonucleotides containing a mutantsequence, flanked by restriction sites enabling ligation to fragments ofthe native sequence. Following ligation, the resulting reconstructedsequence encodes an analog having the desired amino acid insertion,substitution, or deletion.

[0032] Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene whereinpredetermined codons can be altered by substitution, deletion orinsertion. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981);Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methodsin Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462all of which are incorporated by reference.

[0033] Native VESPR polypeptide may be modified to create VESPRderivatives by forming covalent or aggregative conjugates with otherchemical moieties, such as glycosyl groups, lipids, phosphate, acetylgroups and the like. Covalent derivatives of VESPR polypeptides may beprepared by linking the chemical moieties to functional groups on VESPRamino acid side chains or at the N-terminus or C-terminus of a VESPRpolypeptide or the extracellular domain thereof. Other derivatives ofVESPR polypeptides within the scope of this invention include covalentor aggregative conjugates of VESPR polypeptides or its fragments withother proteins or polypeptides, such as by synthesis in recombinantculture as N-terminal or C-terminal fusions. For example, the conjugatemay comprise a signal or leader polypeptide sequence (e.g. the α-factorleader of Saccharomyces) at the N-terminus of a VESPR polypeptide. Thesignal or leader peptide co-translationally or post-translationallydirects transfer of the conjugate from its site of synthesis to a siteinside or outside of the cell membrane or cell wall.

[0034] VESPR polypeptide fusions can comprise peptides added tofacilitate purification and identification of VESPR. Such peptidesinclude, for example, poly-His or the antigenic identification peptidesdescribed in U.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology6:1204, 1988.

[0035] The invention further includes VESPR with or without associatednative-pattern glycosylation. VESPR polypeptide expressed in yeast ormammalian expression systems (e.g., COS-7 cells) may be similar to orsignificantly different from a native VESPR polypeptide in molecularweight and glycosylation pattern, depending upon the choice ofexpression system. Expression of VESPR polypeptides in bacterialexpression systems, such as E. coli, provides non-glycosylatedmolecules.

[0036] Equivalent DNA constructs that encode various additions orsubstitutions of amino acid residues or sequences, or deletions ofterminal or internal residues or sequences not needed for biologicalactivity or binding are encompassed by the invention. For example,N-glycosylation sites in the VESPR extracellular domain can be modifiedto preclude glycosylation, allowing expression of a reduced carbohydrateanalog in mammalian and yeast expression systems. N-glycosylation sitesin eukaryotic polypeptides are characterized by an amino acid tripletAsn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr. Thenative human VESPR protein comprises 24 such triplets, at amino acids86-88, 141-143, 149-151, 241-243, 252-254, 386-388, 407-409, 548-550,553-555, 582-584, 588-590, 591-593, 653, 655, 686-688, 692-694, 715-717,741-743, 771-773, 796-798, 821-823, 871-873, 890-892, 895-897 and920-922 of SEQ ID NO:2. Appropriate substitutions, additions ordeletions to the nucleotide sequence encoding these triplets will resultin prevention of attachment of carbohydrate residues at the Asn sidechain. Alteration of a single nucleotide, chosen so that Asn is replacedby a different amino acid, for example, is sufficient to inactivate anN-glycosylation site. Known procedures for inactivating N-glycosylationsites in proteins include those described in U.S. Pat. No. 5,071,972 andEP 276,846, hereby incorporated by reference.

[0037] In another example, sequences encoding Cys residues that are notessential for biological activity can be altered to cause the Cysresidues to be deleted or replaced with other amino acids, preventingformation of incorrect intramolecular disulfide bridges uponrenaturation. Other equivalents are prepared by modification of adjacentdibasic amino acid residues to enhance expression in yeast systems inwhich KEX2 protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, andLys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Lys-Lys pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents aconservative and preferred approach to inactivating KEX2 sites. Thehuman VESPR contains 11 KEX2 protease processing sites

[0038] Nucleic acid sequences within the scope of the invention includeisolated DNA and RNA sequences that hybridize to the VESPR nucleotidesequences disclosed herein under conditions of moderate or highstringency, and that encode biologically active VESPR. Conditions ofmoderate stringency, as defined by Sambrook et al. Molecular Cloning: ALaboratory Manual, 2 ed. Vol. 1, pp. 101-104, Cold Spring HarborLaboratory Press, (1989), include use of a prewashing solution of 5×SSC,0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization conditions of about 55°C., 5×SSC, overnight. Conditions of high stringency include highertemperatures of hybridization and washing. The skilled artisan willrecognize that the temperature and wash solution salt concentration maybe adjusted as necessary according to factors such as the length of thenucleic acid molecule and the relative amount of A, T/U, C and Gnucleotides.

[0039] Due to the known degeneracy of the genetic code wherein more thanone codon can encode the same amino acid, a DNA sequence may vary fromthat shown in SEQ ID NO:1 and still encode a VESPR polypeptide havingthe amino acid sequence of SEQ ID NO:2. Such variant DNA sequences mayresult from silent mutations (e.g., occurring during PCR amplification),or may be the product of deliberate mutagenesis of a native sequence.

[0040] The invention provides equivalent isolated DNA sequences encodingbiologically active VESPR, selected from: (a) cDNA comprising thenucleotide sequence presented in SEQ ID NO:1; (b) DNA capable ofhybridization to a DNA of (a) under moderately stringent conditions andthat encodes biologically active VESPR polypeptide; (c) DNA that isdegenerate as a result of the genetic code to a DNA defined in (a) or(b) and that encodes biologically active VESPR polypeptide; and (d) DNAcomplementary to the DNA of (a), (b) or (c). VESPR polypeptides encodedby such DNA equivalent sequences are encompassed by the invention.

[0041] DNAs that are equivalents to the DNA sequence of SEQ ID NO:1 willhybridize under moderately and highly stringent conditions to the DNAsequence that encodes polypeptides comprising the sequence of SEQ IDNO:2. Examples of VESPR proteins encoded by such DNA, include, but arenot limited to, VESPR fragments and VESPR proteins comprisinginactivated N-glycosylation site(s), inactivated KEX2 proteaseprocessing site(s), or conservative amino acid substitution(s), asdescribed above. VESPR polypeptides encoded by DNA derived from otherspecies, wherein the DNA will hybridize to the cDNA of SEQ ID NO:1 arealso encompassed.

[0042] Variants possessing the requisite ability to bind semaphorins maybe identified by any suitable assay. Biological activity of VESPRpolypeptides may be determined, for example, by competition for bindingto the receptor binding domain of semaphorins (i.e. competitive bindingassays).

[0043] One type of a competitive binding assay for a VESPR polypeptideuses a radiolabeled, soluble VESPR and intact semaphorin-expressingcells. Instead of intact cells, one could substitute solublesemaphorin:Fc fusion proteins bound to a solid phase through theinteraction of a Protein A, Protein G or an antibody to the semaphorinor Fc portions of the molecule, with the Fc region of the fusionprotein. Another type of competitive binding assay utilizes radiolabeledsoluble semaphorins such as a fusion protein, and intact cellsexpressing VESPR.

[0044] Competitive binding assays can be performed followingconventional methodology. In one embodiment, a soluble VESPR polypeptidecan be made to compete with an immobilized receptor for binding with asoluble semaphorin ligand. For example, a radiolabeled solublesemaphorin ligand can be antagonized by soluble VESPR in an assay forbinding activity against a surface-bound semaphorin receptor.Qualitative results can be obtained by competitive autoradiographicplate binding assays, or Scatchard plots may be utilized to generatequantitative results.

[0045] Alternatively, semaphorin binding proteins, such as VESPR oranti-semaphorin antibodies, can be bound to a solid phase such as acolumn chromatography matrix or a similar substrate suitable foridentifying, separating or purifying cells that express semaphorin ontheir surface. Binding of a semaphorin-binding protein to a solid phasecontacting surface can be accomplished by any means, for example, byconstructing a VESPR:Fc fusion protein and binding such to the solidphase through the interaction of Protein A or Protein G. Various othermeans for fixing proteins to a solid phase are well known in the art andare suitable for use in the present invention. For example, magneticmicrospheres can be coated with VESPR and held in the incubation vesselthrough a magnetic field. Suspensions of cell mixtures containingsemaphorin-expressing cells are contacted with the solid phase that hasVESPR polypeptides thereon. Cells having semaphorin on their surfacebind to the fixed VESPR and unbound cells then are washed away. Thisaffinity-binding method is useful for purifying, screening or separatingsuch semaphorin-expressing cells from solution. Methods of releasingpositively selected cells from the solid phase are known in the art andencompass, for example, the use of enzymes. Such enzymes are preferablynon-toxic and non-injurious to the cells and are preferably directed tocleaving the cell-surface binding partner. In the case ofsemaphorin-VESPR interactions, the enzyme preferably would cleave thesemaphorin, thereby freeing the resulting cell suspension from the“foreign” semaphorin receptor material. The purified cell populationthen may be used to repopulate mature (adult) tissues.

[0046] Alternatively, mixtures of cells suspected of containingsemaphorin-positive cells first can be incubated with biotinylatedVESPR. Incubation periods are typically at least one hour in duration toensure sufficient binding to semaphorin The resulting mixture then ispassed through a column packed with avidin-coated beads, whereby thehigh affinity of biotin for avidin provides the binding of the cell tothe beads. Use of avidin-coated beads is known in the art. See Berenson,et al. J. Cell. Biochem., 10D:239 (1986). Washing unbound material andreleasing the bound cells is performed using conventional methods.

[0047] As described above, VESPR can be used to separate cellsexpressing semaphorin. In an alternative method, VESPR or anextracellular domain or a fragment thereof can be conjugated to adetectable moiety such as ¹²⁵I to detect semaphorin-expressing cells.Radiolabeling with ¹²⁵I can be performed by any of several standardmethodologies that yield a functional ¹²⁵I-VESPR molecule labeled tohigh specific activity. Or an iodinated or biotinylated antibody againstthe semaphorin receptor can be used. Another detectable moiety such asan enzyme that can catalyze a colorimetric or fluorometric reaction,biotin or avidin may be used. Cells to be tested forsemaphorin-expression can be contacted with labeled VESPR polypeptide.After incubation, unbound labeled VESPR is removed and binding ismeasured using the detectable moiety.

[0048] The binding characteristics of VESPR (including variants) mayalso be determined using a conjugated semaphorin (for example,¹²⁵I-semaphorin:Fc) in competition assays similar to those describedabove. In this case, however, intact cells expressing semaphorins boundto a solid substrate are used to measure the extent to which a samplecontaining a putative VESPR variant competes for binding with aconjugated semaphorin.

[0049] Other means of assaying for VESPR include the use of anti-VESPRantibodies, cell lines that proliferate in response to VESPR, orrecombinant cell lines that express semaphorin and proliferate in thepresence of VESPR.

[0050] The VESPR proteins disclosed herein also may be employed tomeasure the biological activity of semaphorin proteins in terms of theirbinding affinity for VESPR. As one example, VESPR polypeptides of thepresent invention may be used in determining whether biological activityis retained after modification of a semaphorin protein (e.g., chemicalmodification, truncation, mutation, etc.). The biological activity of asemaphorin protein thus can be ascertained before it is used in aresearch study, or in the clinic, for example.

[0051] VESPR polypeptides of the present invention find use as reagentsthat may be employed by those conducting “quality assurance” studies,e.g., to monitor shelf life and stability of semaphorin protein underdifferent conditions. To illustrate, VESPR polypeptides may be employedin a binding affinity study to measure the biological activity of ansemaphorin protein that has been stored at different temperatures, orproduced in different cell types. The binding affinity of the modifiedsemaphorin protein for VESPR is compared to that of an unmodifiedsemaphorin protein to detect any adverse impact of the modifications onbiological activity of the semaphorin.

[0052] VESPR polypeptides also find use as carriers for deliveringagents attached thereto to cells expressing semaphorins. As described inexample 7 below, a putative human semaphorin is expressed in cells foundin the placenta, testis, ovary and spleen. VESPR polypeptides can thuscan be used to deliver diagnostic or therapeutic agents to these cells(or to other cell types found to express a semaphorin on a cellsurfaces) in in vitro or in vivo procedures.

[0053] Diagnostic and therapeutic agents that may be attached to a VESPRpolypeptide include, but are not limited to, drugs, toxins,radionuclides, chromophores, enzymes that catalyze a colorimetric orfluorometric reaction, and the like, with the particular agent beingchosen according to the intended application. Examples of drugs includethose used in treating various forms of cancer, e.g., nitrogen mustardssuch as L-phenylalanine nitrogen mustard or cyclophosphamide,intercalating agents such as cis-diaminodichloroplatinum,antimetabolites such as 5-fluorouracil, vinca alkaloids such asvincristine, and antibiotics such as bleomycin, doxorubicin,daunorubicin, and derivatives thereof. Among the toxins are ricin,abrin, diptheria toxin, Pseudomonas aeruginosa exotoxin A, ribosomalinactivating proteins, mycotoxins such as trichothecenes, andderivatives and fragments (e.g., single chains) thereof. Radionuclidessuitable for diagnostic use include, but are not limited to, ¹²³I, ¹³¹I,^(99m)Tc, ¹¹¹In, and ⁷⁶Br. Radionuclides suitable for therapeutic useinclude, but are not limited to, ¹³¹I, ²¹¹At, ⁷⁷Br, ¹⁸⁶Re, ¹⁸⁸Re, ²¹²Pb,²¹²Bi, ¹⁰⁹Pd, ⁶⁴Cu, and ⁶⁷Cu.

[0054] Such agents may be attached to the semaphorin receptor by anysuitable conventional procedure. VESPR, being a protein, comprisesfunctional groups on amino acid side chains that can be reacted withfunctional groups on a desired agent to form covalent bonds, forexample. Alternatively, the protein or agent may be derivatized togenerate or attach a desired reactive functional group. Thederivatization may involve attachment of one of the bifunctionalcoupling reagents available for attaching various molecules to proteins(Pierce Chemical Company, Rockford, Ill.). A number of techniques forradiolabeling proteins are known. Radionuclide metals may be attached tothe receptor by using a suitable bifunctional chelating agent, forexample.

[0055] Conjugates comprising VESPR and a suitable diagnostic ortherapeutic agent (preferably covalently linked) are thus prepared. Theconjugates are administered or otherwise employed in an amountappropriate for the particular application.

[0056] Another use of the VESPR of the present invention is as aresearch tool for studying the role that the receptor, in conjunctionwith semaphorins, may play in immune regulation and viral infection. TheVESPR polypeptides of the present invention also may be employed in invitro assays for detection of semaphorin to which it binds or VESPR, orthe interactions thereof.

[0057] As described in Example 16 semaphorins interact with theirmembrane bound receptors of the present invention to synergize withinterferon and Staphylococcus aureus (type C) (SAC) in the production ofIL-12 from dendritic cells. The use of VESPR and its semaphorin ligandto induce IL-12 production promotes natural killer cell and T cellproduction and induces cytokine production (primarily γ-interferon).IL-12 and IL-12 induced γ interferon production favors Th1 celldifferentiation, and downregulates the production of cytokinesassociated with Th2 cell differentiation. IL-12 is known to act as botha proinflammatory cytokine and an immunomodulator. Thus, a soluble VESPRcan be used to antagonize IL-12 production and downregulate anorganism's Th1 cell differentiation. Similarly, a soluble VESPR can beused to promote production of cytokines associated with Th2 celldifferentiation, thus discouraging proinflammatory activity. Also, VESPRin combination with its semaphorin ligand can be used to boost IL-12production in combination with vaccination for those pathogens againstwhich cellular immunity are effective. In this manner the enhancedamount of IL-12 acts as an adjuvant in the vaccination to induce a morepersistent Th1-type immunological memory.

[0058] Furthermore, it is known that administration of IL-12 to tumorbearing animals results in tumor regression and the establishment of atumor-specific immune response. Thus, using a semaphorin ligand to bindwith VESPR in order to enhance or promote IL-12 can induce a curativeimmune response against aggressive micrometastasizing tumors.

[0059] Additionally, as described in example 18, receptors of thepresent invention bind with their semaphorin ligands to increase CD54expression on monocytes. This observation suggests that thesemaphorin/semaphorin receptor interaction mediate cellular activationthat contributes to the proinflammatory activity typically associatedwith monocyte activation. Such activity includes increased phagocytosis,pinocytosis, nitric oxide production and cytokine production. Toantagonize or reverse the proinflammatory activity resulting from theinteraction between the semaphorin ligand and its membrane boundreceptor, a pharmaceutical composition containing a therapeuticallyeffect amount of a soluble VESPR of the present invention can beadministered parenterally to an organism. The soluble VESPR binds withthe semaphorin ligand thus preventing the ligand from binding with amembrane bound receptor and contributing to the proinflammatoryactivity. A therapeutically effect amount of VESPR is an amountsufficient to antagonize proinflammatory activity.

[0060] Semaphorin ligands binding with VESPR to downregulate expressionof MHC Class II molecules and CD86, a co-stimulatory molecule, ondendritic cells, cultured with GM-CSF and IL-4 (see example 17) suggeststhat the interaction between semaphorin ligands and the receptors of thepresent invention are associated with the immune suppression of maturedendritic cells. To antagonize or reverse the immunosuppression activityresulting from the interaction between the semaphorin ligand and itsmembrane bound receptor, a pharmaceutical composition containing atherapeutically effective amount of a soluble VESPR of the presentinvention can be administered parenterally to an organism. The solubleVESPR binds with the semaphorin ligand thus preventing the ligand frombinding with a membrane bound receptor and contributing to theimmunosuppression activity. Alternatively, in patients or organisms thatsuffer from the effects of chronic inflammation, administeringappropriate semaphorin ligands will contribute to suppressing theproinflammatory activity of differentiated macrophages.

[0061] VESPR polypeptides of the invention can be formulated accordingto known methods used to prepare pharmaceutically useful compositions.VESPR can be combined in admixture, either as the sole active materialor with other known active materials, with pharmaceutically suitablediluents (e.g., Tris-HCl, acetate, phosphate), preservatives (e.g.,Thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers,adjuvants and/or carriers. Suitable carriers and their formulations aredescribed in Remington's Pharmaceutical Sciences, 16th ed. 1980, MackPublishing Co. In addition, such compositions can contain VESPRpolypeptide complexed with polyethylene glycol (PEG), metal ions, orincorporated into polymeric compounds such as polyacetic acid,polyglycolic acid, hydrogels, etc., or incorporated into liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts or spheroblasts. Such compositions will influence thephysical state, solubility, stability, rate of in vivo release, and rateof in vivo clearance of VESPR. VESPR polypeptide can also be conjugatedto antibodies against tissue-specific receptors, ligands or antigens, orcoupled to ligands of tissue-specific receptors.

[0062] VESPR polypeptides can be administered topically, parenterally,or by inhalation. The term “parenteral” includes subcutaneousinjections, intravenous, intramuscular, intracisternal injection, orinfusion techniques. These compositions will typically contain aneffective amount of the VESPR, alone or in combination with an effectiveamount of any other active material. Such dosages and desired drugconcentrations contained in the compositions may vary depending uponmany factors, including the intended use, patient's body weight and age,and route of administration. Preliminary doses can be determinedaccording to animal tests, and the scaling of dosages for humanadministration can be performed according to art-accepted practices.

[0063] VESPR polypeptides may exist as oligomers, such ascovalently-linked or non-covalently-linked dimers or trimers. Oligomersmay be linked by disulfide bonds formed between cysteine residues ondifferent VESPR molecules. In one embodiment of the invention, a VESPRdimer is created by fusing VESPR to the Fc region of an antibody (e.g.,IgG1) in a manner that does not interfere with binding of VESPR to asemaphorin ligand-binding domain. The Fc polypeptide preferably is fusedto the C-terminus of a soluble VESPR (comprising only the ligand-bindingdomain). General preparation of fusion proteins comprising heterologouspolypeptides fused to various portions of antibody-derived polypeptides(including the Fc domain) has been described, e.g., by Ashkenazi et al.(PNAS USA 88:10535, 1991) and Byrn et al. (Nature 344:677, 1990), herebyincorporated by reference. A gene fusion encoding the VESPR:Fc fusionprotein is inserted into an appropriate expression vector. VESPR:Fcfusion proteins are allowed to assemble much like antibody molecules,whereupon interchain disulfide bonds form between Fc polypeptides,yielding divalent. If fusion proteins are made with both heavy and lightchains of an antibody, it is possible to form a VESPR oligomer with asmany as four VESPR extracellular regions. Alternatively, one can linktwo soluble VESPR domains with a peptide linker.

[0064] Suitable host cells for expression of VESPR polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in Pouwels et al. CloningVectors: A Laboratory Manual, Elsevier, N.Y., (1985). Cell-freetranslation systems could also be employed to produce VESPR polypeptidesusing RNAs derived from DNA constructs disclosed herein.

[0065] Prokaryotes include gram negative or gram positive organisms, forexample, E. coli or Bacillus. Suitable prokaryotic host cells fortransformation include, for example, E. coli, Bacillus subtilis,Salmonella typhimurium, and various other species within the generaPseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic hostcell, such as E. coli, a VESPR polypeptide may include an N-terminalmethionine residue to facilitate expression of the recombinantpolypeptide in the prokaryotic host cell. The N-terminal methionine maybe cleaved from the expressed recombinant VESPR polypeptide.

[0066] VESPR polypeptides may be expressed in yeast host cells,preferably from the Saccharomyces genus (e.g., S. cerevisiae). Othergenera of yeast, such as Pichia, K. lactis or Kluyveromyces, may also beemployed. Yeast vectors will often contain an origin of replicationsequence from a 2μ yeast plasmid, an autonomously replicating sequence(ARS), a promoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J.Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900,1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in Hitzeman, EPA-73,657 or in Fleer et.al., Gene, 107:285-195 (1991); and van den Berg et. al., Bio/Technology,8:135-139 (1990). Another alternative is the glucose-repressible ADH2promoter described by Russell et al. (J. Biol. Chem. 258:2674, 1982) andBeier et al. (Nature 300:724, 1982). Shuttle vectors replicable in bothyeast and E. coli may be constructed by inserting DNA sequences frompBR322 for selection and replication in E. coli (Amp^(r) gene and originof replication) into the above-described yeast vectors.

[0067] The yeast α-factor leader sequence may be employed to directsecretion of the VESPR polypeptide. The α-factor leader sequence isoften inserted between the promoter sequence and the structural genesequence. See, e.g., Kurjan et al., Cell 30:933, 1982; Bitter et al.,Proc. Natl. Acad. Sci. USA 81:5330, 1984; U.S. Pat. No. 4,546,082; andEP 324,274. Other leader sequences suitable for facilitating secretionof recombinant polypeptides from yeast hosts are known to those of skillin the art. A leader sequence may be modified near its 3′ end to containone or more restriction sites. This will facilitate fusion of the leadersequence to the structural gene.

[0068] Yeast transformation protocols are known to those of skill in theart. One such protocol is described by Hinnen et al., Proc. Natl. Acad.Sci. USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 μg/ml adenine and 20 μg/ml uracil.

[0069] Yeast host cells transformed by vectors containing ADH2 promotersequence may be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Depression of the ADH2 promoter occurs when glucose is exhaustedfrom the medium.

[0070] Mammalian or insect host cell culture systems could also beemployed to express recombinant VESPR polypeptides. Baculovirus systemsfor production of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6:47 (1988). Established cell linesof mammalian origin also may be employed. Examples of suitable mammalianhost cell lines include the COS-7 line of monkey kidney cells (ATCC CRL1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, andBHK (ATCC CRL 10) cell lines, and the CV-1/EBNA-1 cell line derived fromthe African green monkey kidney cell line CVI (ATCC CCL 70) as describedby McMahan et al. (EMBO J. 10: 2821, 1991).

[0071] Transcriptional and translational control sequences for mammalianhost cell expression vectors may be excised from viral genomes. Commonlyused promoter sequences and enhancer sequences are derived from Polyomavirus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites may be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment which may also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40fragments may also be used, provided the approximately 250 bp sequenceextending from the Hind III site toward the Bgl I site located in theSV40 viral origin of replication site is included.

[0072] Exemplary expression vectors for use in mammalian host cells canbe constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280,1983). A useful system for stable high level expression of mammaliancDNAs in C127 murine mammary epithelial cells can be constructedsubstantially as described by Cosman et al. (Mol. Immunol. 23:935,1986). A useful high expression vector, PMLSV N1/N4, described by Cosmanet al., Nature 312:768, 1984 has been deposited as ATCC 39890.Additional useful mammalian expression vectors are described inEP-A-0367566, and in U.S. patent application Ser. No. 07/701,415, filedMay 16, 1991, incorporated by reference herein. The vectors may bederived from retroviruses. In place of the native signal sequence, andin addition to an initiator methionine, a heterologous signal sequencemay be added, such as the signal sequence for IL-7 described in U.S.Pat. No. 4,965,195; the signal sequence for IL-2 receptor described inCosman et al., Nature 312:768 (1984); the IL-4 signal peptide describedin EP 367,566; the type I IL-1 receptor signal peptide described in U.S.Pat. No. 4,968,607; and the type II IL-1 receptor signal peptidedescribed in EP 460,846.

[0073] VESPR polypeptides as isolated, purified or homogeneous proteinsaccording to the invention may be produced by recombinant expressionsystems as described above or purified from naturally occurring cells.VESPR can be purified to substantial homogeneity, as indicated by asingle protein band upon analysis by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE).

[0074] One process for producing VESPR comprises culturing a host celltransformed with an expression vector comprising a DNA sequence thatencodes VESPR polypeptide under conditions sufficient to promoteexpression of VESPR polypeptide. The receptor is then recovered fromculture medium or cell extracts, depending upon the expression systememployed. As is known to the skilled artisan, procedures for purifying arecombinant protein will vary according to such factors as the type ofhost cells employed and whether or not the recombinant protein issecreted into the culture medium.

[0075] For example, when expression systems that secrete the recombinantprotein are employed, the culture medium first may be concentrated usinga commercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a purificationmatrix such as a gel filtration medium. Alternatively, an anion exchangeresin can be employed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,(e.g., silica gel having pendant methyl or other aliphatic groups) canbe employed to further purify VESPR polypeptide. Some or all of theforegoing purification steps, in various combinations, are well knownand can be employed to provide a substantially homogeneous recombinantprotein.

[0076] It is possible to utilize an affinity column comprising thereceptor-binding domain of a semaphorin that binds VESPR toaffinity-purify expressed VESPR polypeptides. VESPR polypeptides can beremoved from an affinity column using conventional techniques, e.g., ina high salt elution buffer and then dialyzed into a lower salt bufferfor use or by changing pH or other components depending on the affinitymatrix utilized. Alternatively, the affinity column may comprise anantibody that binds VESPR. Example 20 describes a procedure foremploying VESPR of the invention to generate monoclonal antibodiesdirected against VESPR

[0077] Recombinant protein produced in bacterial culture can be isolatedby initial disruption of the host cells, centrifugation, extraction fromcell pellets if an insoluble polypeptide, or from the supernatant fluidif a soluble polypeptide, followed by one or more concentration,salting-out, ion exchange, affinity purification or size exclusionchromatography steps. Finally, RP-HPLC can be employed for finalpurification steps. Microbial cells can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

[0078] Transformed yeast host cells are preferably employed to expressVESPR as a secreted polypeptide in order to simplify purification.Secreted recombinant polypeptide from a yeast host cell fermentation canbe purified by methods analogous to those disclosed by Urdal et al. (J.Chromatog. 296:171, 1984). Urdal et al. describe two sequential,reversed-phase HPLC steps for purification of recombinant human IL-2 ona preparative HPLC column.

[0079] Useful fragments of the VESPR nucleic acids include antisense orsense oligonucleotides comprising a single-stranded nucleic acidsequence (either RNA or DNA) capable of binding to target VESPR mRNA(sense) or VESPR DNA (antisense) sequences. Antisense or senseoligonucleotides, according to the present invention, comprise afragment of the coding region of VESPR cDNA. Such a fragment generallycomprises at least about 14 nucleotides, preferably from about 14 toabout 30 nucleotides. The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988)and van der Krol et al. (BioTechniques 6:958, 1988).

[0080] Binding of antisense or sense oligonucleotides to target nucleicacid sequences results in the formation of duplexes that blocktranscription or translation of the target sequence by one of severalmeans, including enhanced degradation of the duplexes, prematuretermination of transcription or translation, or by other means. Theantisense oligonucleotides thus may be used to block expression of VESPRproteins. Antisense or sense oligonucleotides further compriseoligonucleotides having modified sugar-phosphodiester backbones (orother sugar linkages, such as those described in WO91/06629) and whereinsuch sugar linkages are resistant to endogenous nucleases. Sucholigonucleotides with resistant sugar linkages are stable in vivo (i.e.,capable of resisting enzymatic degradation) but retain sequencespecificity to be able to bind to target nucleotide sequences. Otherexamples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10448, and other moieties that increasesaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

[0081] Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. Antisense or sense oligonucleotides are preferably introducedinto a cell containing the target nucleic acid sequence by insertion ofthe antisense or sense oligonucleotide into a suitable retroviralvector, then contacting the cell with the retrovirus vector containingthe inserted sequence, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see PCT Application US90/02656).

[0082] Sense or antisense oligonucleotides also may be introduced into acell containing the target nucleotide sequence by formation of aconjugate with a ligand binding molecule, as described in WO 91/04753.Suitable ligand binding molecules include, but are not limited to, cellsurface receptors, growth factors, other cytokines, or other ligandsthat bind to cell surface receptors. Preferably, conjugation of theligand binding molecule does not substantially interfere with theability of the ligand binding molecule to bind to its correspondingmolecule or receptor, or block entry of the sense or antisenseoligonucleotide or its conjugated version into the cell.

[0083] Alternatively, a sense or an antisense oligonucleotide may beintroduced into a cell containing the target nucleic acid sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. The sense or antisense oligonucleotide-lipid complex ispreferably dissociated within the cell by an endogenous lipase.

[0084] In addition to the above, the following examples are provided toillustrate particular embodiments and not to limit the scope of theinvention.

EXAMPLE 1 Preparing an Ectromelia Semaphorin/Fc Fusion Protein

[0085] The following describes preparation of an Ectromelia SemaphorinA39R/immunoglobulin fusion protein (A39R/Fc). The process includedpreparing a DNA construct that encodes the fusion protein, transfectinga cell line with the DNA construct, and harvesting supernatants from thetransfected cells. The A39R/Fc fusion protein was used as described inExamples 3, 4, 5 and 6 to study VESPR binding characteristics andisolate VESPR.

[0086] DNA encoding A39R semaphorin was isolated and amplified fromgenomic Ectromelia virus DNA using PCR techniques and synthesizedoligonucleotide primers whose sequences were based on published A39Rsequences in the Copenhagen strain of Vaccinia Virus. The Copenhagenstrain A39R DNA sequence is described in Goebel, S. J. et al. Virology179:247, 1990. The isolated Ectromelia A39R DNA is presented in SEQ IDNO:7 and the protein encoded by the DNA is presented in SEQ ID NO:8. Theupstream oligonucleotide primer introduced an Spe1 site upstream ofamino acid 15 of the A39R polypeptide. A downstream oligonucleotideprimer introduced a Not1 site downstream of the termination codon ofEctromelia A39R, after amino acid 399. The primer sequences were asfollows: Upstream Spe1 primer: Upstream Spe1 primer:TGTCACTAGT ATCGAATGGC ATAAGTTTGA A (SEQ ID NO:3)      Spe1        A39RDNA Downstream Not1 primer: GACAGCGGCC GC CTATTACA TTTTAAGTAT TTT (SEQID NO:4)      Not1           A39R DNA

[0087] A Bgl II to Nsl I restriction fragment containing a mutein humanFc region of immunoglobulin as described by Baum et al. Cir. Sh. 44:30(1994) was ligated into an expression vector (pDC304) containing amurine IL-7 signal peptide and a FLAG™ octapeptide as described in U.S.Pat. No. 5,011,912. The PCR amplified DNA encoding amino acids 15-399 ofEctromelia A39R was then ligated into the expression vector containingthe mutein human Fc region, the murine IL-7 signal peptide and FLAG™peptide, in a two way ligation. The resulting DNA construct wastransfected into the monkey kidney cell lines CV-1/EBNA (withco-transfection of psv3neo). After 7 days of culture in mediumcontaining 0.5% low immunoglobulin bovine serum, a solution of 0.2%azide was added to the supernatant and the supernatant was filteredthrough a 0.22 μm filter. Then approximately 1 L of culture supernatantwas passed through a BioCad Protein A HPLC protein purification systemusing a 4.6×100 mm Protein A column (POROS 20A from PerSeptiveBiosystems) at 10 mL/min. The Protein A column binds the Fc Portion ofthe fusion protein in the supernatant, immobilizing the fusion proteinand allowing other components of the supernatant to pass through thecolumn. The column was washed with 30 mL of PBS solution and boundfusion protein was eluted from the HPLC column with citric acid adjustedto pH 3.0. Eluted purified fusion protein was neutralized as it elutedusing 1M HEPES solution at pH 7.4.

EXAMPLE 2 Preparing an Ectromelia Semaphorin/polyHis Fusion Protein

[0088] The following describes preparation of an Ectromelia A39R/polyHisfusion protein (A39R/polyHis). The process included preparing a DNAconstruct that encodes the fusion protein, transfecting a cell line withthe DNA construct, and harvesting supernatants from the transfectedcells.

[0089] DNA encoding Ectromelia A39R (amino acids 1-399 of A39R ORF, SEQID NO:8) was isolated and amplified from genomic Ectromelia virus DNAusing PCR techniques and synthesized oligonucleotide primers. Theprimers added a Not 1 site at the 5′ end and the motif Gly-Ser-6×HIS atthe 3′ end for use in purification processes. After the Gly-Ser-6×HISmotif the primers added an in-frame termination codon and a Bgl 2 site.The PCR product was cut and cloned in pDC409 expression vector (McMahonet al., EMBO J. 10:2821,1991).

[0090] The resulting DNA construct was transiently transfected into themonkey cell line COS-1 (ATCC CRL-1650). Following a 7 day culture inmedium containing 0.5% low immunoglobulin bovine serum, cellsupernatants were harvested and a solution of 0.2% sodium azide wasadded to the supernatants. The supernatants were filtered through a 0.22μm filter, concentrated 10 fold with a prep scale concentrator(Millipore; Bedford, Mass.) and purified on a BioCad HPLC proteinpurification equipped with a Nickel NTA Superflow self pack resin column(Qiagen, Santa Clarita, Calif.). After the supernatant passed throughthe column, the column was washed with Buffer A (20 mM NaPO4, pH 7.4;300 mMNaCl; 50 mM Imidazole). Bound protein was then eluted from thecolumn using a gradient elution techniques. Fractions containing proteinwere collected and analyzed on a 4-20% SDS-PAGE reducing gel. Peakscontaining A39R/polyHis fusion protein were pooled, concentrated 2 fold,and then dialyzed in PBS. The resulting A39R/polyHis fusion protein wasthen filtered through a 0.22 μm sterile filter.

EXAMPLE 3 Screening Cell Lines for Binding to A39R

[0091] The A39R/Fc fusion protein prepared as described in Example 1 wasused to screen cell lines for binding using quantitative binding studiesaccording to standard flow cytometry methodologies. For each cell linescreened, the procedure involved incubating approximately 100,000 of thecells blocked with 2% FCS (fetal calf serum), 5% normal goat serum and5% rabbit serum in PBS for 1 hour. Then the blocked cells were incubatedwith 5 μg/mL of A39R/Fc fusion protein in 2% FCS, 5% goat serum and 5%rabbit serum in PBS. Following the incubation the sample was washed 2times with FACS buffer (2% FCS in PBS) and then treated with mouse antihuman Fc/biotin (purchased from Jackson Research) and SAPE(streptavidin-phycoerythrin purchased from Molecular Probes). Thistreatment causes the antihuman Fc/biotin to bind to any bound A39R/Fcand the SAPE to bind to the anti-human Fc/biotin resulting in afluorescent identifying label on A39R/Fc which is bound to cells. Thecells were analyzed for any bound protein using fluorescent detectionflow cytometry. The results indicated the A39R semaphorin binds well tohuman NK cells, murine splenic B cells, human PB T cells, human T, B,erythroid, lymphoid and myeloid precursor cells, fibroblasts andepithelial lineage. Table I details the results of the flow cytometrystudies. A “+” indicates that binding was detected between the cellsurface and A39R. A “−” indicates that no binding was detected betweenthe cell surface and A39R. TABLE I A39R Binding Cell Line Result Namalwa(B cell-like lymphoma - human) + CB23 (Human Cord Blood B Cell Line) +EU-1 (Human pre B Cell Line) + MP-1 (Human B Cell Lymphoma) + PB B(Human Peripheral Blood B Cells) + Mouse Splenic B Cells + Mouse SplenicB Cells + CD40L + U937 (Human Monocyte-Type Cell) + HSB2 (Human T CellLine) + K299 (Non Hodgkin's Lymphoma) + TE71 (Mouse Thymic Epithelium) +IEC18 (Rat Intestinal Epithelium) + IMTLH (Human Bone Marrow DerivedStroma) + W126 (Human Lung Epithelium) + PL-1 (Human T-Cell Clone + VK-1Human T-Cell Clone + Primary Peripheral Blood T Cells + Primary Human NKCells + RAJI (Burkitt's Lymphoma) − KG1 (Human myeloid Cell Line) −THP-1 (Human Promonocytic Cell Line) + MC6 (Mouse Mast Cell) − EL4(Mouse Thymoma) − BeWo (Chorio Carcinoma) − Primary Mouse DendriticCells + Primary Human Dendritic Cells +

EXAMPLE 4 Identifying a Putative Semaphorin Receptor

[0092] CB23 cells (human cord blood B cell line) and human PB T cellsthat tested positive for binding to A39R were tested for expression of aputative receptor and to determine if any receptor is expressed as amembrane bound molecule, soluble molecule, or both. Broadly, theanalyses involved radiolabeling CB23 and human PB T cell surfaces,harvesting and treating cell supernatants and lysates with an A39R/Fcfusion protein to precipitate any putative receptor, and thenvisualizing an immunoprecipitate on an electrophoretic gel.

[0093] In particular, the procedure involved first radiolabelingapproximately 1×10⁷ CB23 or PB T cells with [¹²⁵I] as described byBenjamin et al.; Blood 75,:2017-2023 (1990). Cultured cell supernatantswere harvested and clarified by centrifugation at 14,000 rpm for 30minutes. Cell lysates were generated by incubating the cells on ice for30 minutes in 1 mm L phosphate-buffered saline with 1% Triton-X 100containing protease inhibitors phenylmethylsulfonyl fluoride,Pepstatin-A, and Leupeptin. The lysates were clarified by centrifugationat 14,000 rpm for 30 minutes. In order to precipitate any receptorpresent in the lysate and/or supernatant, 200 μL of the cell supernatantor lysate was incubated with 2 μg of A39R/Fc fusion protein prepared asdescribed in Example 1. The incubation was carried out for 1 hour withgentle rocking at 4° C. An Fc protein control sample was prepared andincubation in the same manner. Following the incubation, Protein-ASepharose beads (#17-0780-01 Pharmacia Biotech Inc., Piscataway, N.J.)were added to the lysates and supernatants and the mixture was incubatedfor 1 hour with gentle rocking at 4° C. The beads were washedextensively with a PBS 1% Triton-X 100 solution. Bound protein waseluted and analyzed by SDS PAGE. Protein bands were visualized byautoradiography and a single, approximately 200K Da band was found tobind to A39R/Fc but not to the control Fc Protein. The semaphorinreceptor was present in cell lysate and cell supernatant, confirming itsexpression as membrane bound protein and as a secreted soluble protein.

EXAMPLE 5 Isolating and Sequencing a Semaphorin Receptor

[0094] The A39R/Fc fusion protein, prepared as described in Example 1,was used to isolate a human semaphorin receptor polypeptide and aprocedure for the isolated polypeptide purification was confirmed. Thesemaphorin receptor was isolated by suspending CB23 cell pellets in asolution of protease inhibitors that included 1 mM each of PMSF,Leupeptin, Aprotinin, Pepstatin A, 10 μg/mL APMSF, and 1 mM EDTA inhomogenization buffer (10 mM phosphate, 30 mM NaCl, pH 7.4). The cellswere dounce homogenized and layered over a solution of 41% sucrose inhomogenization buffer and the spun down in a Beckman SW-28 rotor at25,000 rpm, at 4° C. for 45 minutes. The interphases were collected anddiluted in cold homogenization buffer, dounced, and spun. The resultingclean membrane pellets were stored at −80° C.

[0095] Membrane pellets prepared from 240 mLs of packed cells werecombined with 100 mLs of an aqueous solution of 20 mM Tris, 150 mM NaCl,the protease inhibitors identified above, 1% Triton X-100 and 0.1 mM ofCaCl₂, MgCl₂, and McCl₂ salts (Buffer A). The suspended pellets weredounced and spun in a SW-28 rotor for 30 minutes at 25,000 rpm at 4° C.The supernatant was placed onto a 100 mL wheat germ agglutinin columnand allowed to elute at a rate of 1 mL/minute with 10 column volumes ofBuffer A. Proteins that were specifically bound to the column were theneluted with Buffer A containing 0.2 M N-acetyl glucosamine.

[0096] Fractions testing positive for protein were pooled and incubatedwith 100 μg of A39R/Fc fusion protein for 1 hour at 4° C. The incubatedmixture was run through a sepharose column to remove material that didnot specifically bind and then allowed to pass through a 0.5 mL columnof Protein A/Sepharose solid support. The Protein A/Sepharose solidsupport was washed with 20 column volumes of PBS containing 1% TritonX-100 followed by a wash with PBS to wash off any unbound material. Thenproteins that were retained on the Protein A/Sepharose column wereeluted in a stepwise manner with 0.35 mL fractions of 50 mM citrate atpH 3.0. Fractions that tested positive for protein were combined andconcentrated to 50 μL using a 10 kD MWCO Centricon concentrator. Proteinin the resulting concentrated sample were reduced and then alkylatedusing standard DTT and iodoacetic acid procedures. The alkylationproteins were then electrophoresed on an 8% gel. Proteins on the gelwere visualized with coomassie-G in 50% MeOH containing 5% acetic acidand then destained in 50% MeOH.

[0097] The approximately 200 kD band, located by comparison to proteinstandards, was excised with a razor blade and washed overnight in 100 mMammonium carbonate. The gel slice was speed evaporated until dry and a1:10 solution of trypsin in 100 mM ammonium carbonate was added to thedried slide. The slide was incubated at 37° C. for 16 hours and thenprotein in the slice was extracted with 50% acetonitrile with 5% formicacid three times while incubating 30 minutes with each extraction.

[0098] The trypsin digested peptide fragments were lyophilized,reconstituted in 50 μL of 0.1% trifluoroacetic acid, and separated byRP-HPLC on a 500 μid×25 cm capillary column packed with C-18 reversephase packing. The HPLC liquid phase was an acetonitrile/water gradientof 10% after 5 minutes, 85% after 105 minutes. Eluting protein wasdetected at 215 nm. Each protein was collected as it eluted in separatefractions and N-terminal sequence analysis of the peptides in thefraction was performed on a 494 Procise sequencer according to themanufacturer's instruction.

[0099] RP-HPLC fractions, obtained as described above, were dried on avacuum centrifuge and peptides in the fraction were dissolved in 6 μL of50% methanol containing 0.5% acetic acid. Two microliters (2 μL) of eachof the peptide solutions were loaded into nanospray tips (ProteinAnalysis Company, Odense, Denmark). Data were obtained with a FinniganTSQ700 triple quadrupole mass spectrometer (San Jose, Calif.) equippedwith a nanospray source. Mass spectra were acquired at unit resolution.For tandem mass spectrometry, the first quadrupole was operated at aresolution sufficient to pass a 3-4 Da wide window, and the thirdquadrupole was operated at unit resolution. Collision gas was suppliedat a pressure of 4 mTorr. Methyl esterification was performed usingstandard esterification procedures.

[0100] The tandem mass spectrometry analysis of the trypsin generatedpeptides provided amino acid sequence information for isolated portionsof the purified protein. The tandem mass spectral data were used incomputer assisted screening of non-redundant protein databases and ESTdatabases using the local SEQUEST algorithm search tool (Eng, J. K etal. J am Soc. Mass 1994). The peptide query sequencesGluGluThrProValPheTyrLys corresponding to amino acids 421-428 of SEQ IDNO:2, and AsnIleTyrIleTyrLeuThrAlaGlyLys, corresponding to amino acids436-445 identified EST No. 248534 (Accession N78220) as containingpeptide sequences having 100% identity to the query peptide sequences.The peptide query sequence ThrValLeuPheLeuGlyThrGlyAspGlyGlnLeuLeuLyscorresponding to amino acids 388-401 identified EST No. R08946 ascontaining a 100% identity to the query.

[0101] The 100% identity between portions of EST 248534 and threepeptide fragments of the purified protein strongly suggested that thecDNA contained within EST 248534 represented a portion of the nucleotidesequence for the coding region of the purified protein. A source ofsemaphorin receptor cDNA was identified using phage library screeningmethods and PCR primers based upon EST 248534.

[0102] The oligonucleotide primers had the following nucleotidesequences: ATCGCATCAT CTACCTTCAT CCATTCCGAC CTG (SEQ ID NO:9) TAAACACTCCGAACAGGATT TATGTTTATT GCA (SEQ ID NO:10)

[0103] PCR isolation and amplification methodologies were carried outusing a panel of human tissue cDNA phage libraries as templates for thePCR reactions. The PCR reaction mixture included 1 μL of phage librarystock, PCR oligonucleotide primers at 0.3 μM final concentration, 1×PC2buffer (Ab Peptides, Inc., St. Louis, Mo.), 0.2 mM each of dATP, dCTP,dGTP, dTTP (Pharmacia Biotech) 0.2 μL of a 16:1 mix Klen-Taq/Ventpolymerase (Klen-Taq polymerase, Ab Peptides, Inc. and Vent polymerase,New England Biolabs, Beverly, Mass.) in a 30 μL final reaction volume.The PCR reaction cycles included one cycle at 98° C. for 5 minutes;thirty cycles at 98° C. for 45 seconds, thirty cycles at 68° C. for 45seconds; thirty cycles at 72° C. for 45 seconds, and 1 cycle at 72° C.for 5 minutes using a Robocycler 96 from Stratagene, La Jolla, Calif.cDNAs in several libraries were positively identified as containing DNAencoding the purified VESPR protein based upon the appearance of anappropriate sized DNA band in electrophoresed PCR product.

[0104] Two of the phage libraries, human foreskin fibroblast and humandermal fibroblasts, were chosen for additional analysis. Libraries wereplated according to established prodedures and probed with aradiolabeled random primer probe derived from a PCR amplificationproduct using EST 248534 as a template. The PCR conditions used toobtained the amplification product were as described above and the probewas generated using Prime-IT II Random Primer Labeling Kit fromStratagene, La Jolla, Calif. Approximately 1×10⁶ cpm/mL of purifiedprobe was used to probe human foreskin phage libraries on nylon membranefilters overnight at 63° C. in a hybridization buffer of 10×Denhardtssolution, 50 mM Tris at pH 7.5, 0.9 M NaCl, 0.01% Sodium Pyrophosphate,1% sodium dodecyl sulfate, and 200 μg/mL denatured, fragmented salmonsperm DNA. After probing, the probed membranes were washed once in6×SSC, 0.1% SDS for 20 minutes, once in 2×SSC, 0.1% SDS for 20 minutes,once in 1×SSC, 0.1% SDS for 20 minutes, and once in 0.1×SSC, 0.1% SDSfor 20 minutes at 63° C. The probed and washed filters were exposed toX-omat AR X-ray film (Eastman-Kodak Corp.) overnight. Four overlappingcDNAs were identified. The overlapping cDNAs, together with thesequenced trypsin digest generated protein fragments were used tocomplete and confirm the coding sequence of VESPR as shown in SEQ IDNO:1 and the amino acid sequence presented in SEQ ID NO:2.

EXAMPLE 6 Monoclonal Antibodies to A39R Semaphorin

[0105] This example illustrates a method for preparing antibodies toA39R semaphorin. Purified A39R/Fc was prepared as described in Example 1above. The purified protein was used to generate antibodies against A39Rsemaphorin as described in U.S. Pat. No. 4,411,993. Briefly, mice wereimmunized at 0, 2 and 6 weeks with 10 μg with A39R/Fc. The primaryimmunization was prepared with TITERMAX adjuvant, from Vaxcell, Inc.,and subsequent immunizations were prepared with incomplete Freund'sadjuvant (IFA). At 11 weeks, the mice were IV boosted with 3-4 μgA39R/Fc in PBS. Three days after the IV boost, splenocytes wereharvested and fused with an Ag8.653 myeloma fusion partner using 50%aqueous PEG 1500 solution. Hybridoma supernatants were screened for A39Rantibodies by dot blot assay against A39R/FC and an irrelevant Fcprotein.

EXAMPLE 7 Northern Blot Analyses for Tissue Expressing SemaphorinReceptor

[0106] The following describes Northern Blot experiments carried out toidentify tissue and cell types that express VESPR polypeptides of thepresent invention. The results confirm the cell binding results obtainedusing flow cytometry analysis and the A39R/Fc fusion protein.

[0107] As described in Example 5, EST data base searches resulted in thediscovery of an EST that was believed to be a partial clone of the VESPRof SEQ ID NO:2 (EST 248534). A riboprobe template was generated usingPCR techniques and oligonucleotide primers that were based onnucleotides 1-372 of EST 248534. The upstream and down stream primersthat encompasses nucleotides 1-372 of EST 248534 had the followingsequences: (SEQ ID NO:5) GCGGGACTCA GAGTCACC (SEQ ID NO:6)GGATCCTAAT ACGACTCACT ATAGGGAGGA AACCACTCCG AAC

[0108] The underlined portion is a T7 site.

[0109] The two primers were used to isolate and amplify a PCR productfrom EST248534 for use in generating a riboprobe. The riboprobe wasgenerated using Ambion's MAXIscript SP6/T7 kit by combining 3 μL ofRNAse free water, 2 μL 10×transcription buffer, 1 μL each of 10 mMdATP/dCTP/dGTP, 5 μL 5′/3′ EST 248534 PCR Product, 5 μL Amersham[α³²P]UTP 10 mCi/mL, 2 μL T7 RNA polymerase at room temperature. Thecombination was microfuged, spun briefly, and incubated at 37° C. for 30minutes. Then 1 μL DNAse was added to the mixture and allowed to reactfor 15 minutes at 37° C. The reaction product was passed through twocolumn volumes of G-25 packing (Boehringer). One microliter (1 μL) ofthe riboprobe was counted in a scintillation counter for 1 minute todetermine cpm/mL

[0110] Northern blots were generated by fractionating polyadenylated RNAfrom a variety of cell lines on a 1.2% agarose formaldehyde gel andblotting the RNA onto Hybond Nylon membranes (Amersham, ArlingtonHeights, Ill.). Standard northern blot generating procedures asdescribed in Maniatis, (Molecular Cloning: a Laboratory Manual, ColdSpring Harbor Lab. Press, 1989) were used. Total RNA multiple tissuenorthern blots were purchased commercially (BioChain Institute, Inc.,San Leandro, Calif. Cat #s 021001, 021002, 021003).

[0111] The Northern blots were prehybridized in a 50% formamidehybridization solution (30 mL 20×SSC, 2 mL 100×Denhardt's reagent, 1 mLof 10 mg/mL denatured fragmented salmon sperm DNA, 50 mL 100% formamideand 20 mL 10% SDS. The total RNA blots were pre-hybridized at 42° C. for4 hours and the polyA+RNA blots were pre-hybridized at 63° C. for 4hours. The riboprobe was added to clean hybridization solution (same asprehybridization solution) at a count of 10⁶ cpm/mL. Theprehybridization solution was removed from the blots and thehybridization solution and riboprobe were added to the blots. Thehybridization was allowed to proceed overnight with gentle shaking. Thetotal RNA blots hybridized at 63° C. and the polyA+RNA blots hybridizedat 63° C.

[0112] The probed total RNA blots were washed once for 30 minutes in2×SSC containing 0.05% SDS at 42° C. and once for 30 minutes in 2×SSCcontaining 0.05% SDS at 55° C.; twice for 30 minutes in 0.1×SSCcontaining 0.1% SDS at 63° C.; three times for 30 minutes in 0.1×SSCcontaining 0.1%SDS and then exposed to X-ray film. The poly A+blots werewashed once for 30 minutes in a 2×SSC solution containing 0.05% SDS at63° C. and once for 30 minutes in 1×SSC containing 0.1% SDS and thenexposed to x-ray film.

[0113] The results of probing the Northern blots and visualizing theresulting x-ray film for positively binding probes confirm that VESPR isexpressed in the same cells as those that showed positive binding inflow cytometry experiments. Hybridizing RNA was detected in MP-1, HFFand CB23 cells. Primary tissues showing positive RNA included heart,brain, lung, spleen and placenta. No RNA was detecting in RAJ1 cells.

EXAMPLE 8 Generating AHV Semaphorin Fc Fusion Protein

[0114] The following describes preparing an AHVSemaphorin/immunoglobulin fusion protein (AHVSema/Fc). The processincluded preparing a DNA construct that encodes the fusion protein,transfecting a cell line with the DNA construct, and harvestingsupernatants from the transfected cells.

[0115] DNA encoding AHV-Sema is described in Ensser et al. J. Gen. Vir.76:1063-1067, 1995. DNA encoding AHV-Sema amino acids 70-653 wasisolated and amplified from Alcelaphine herpesvirus DNA strain WC11(Plowright, W. et al. Nature 188:1167-1169, 1960) using PCR techniquesand synthesized oligonucleotide primers whose sequences were based onthe published AHV-Sema sequence. The upstream oligonucleotide primerintroduced a Spe 1 site. A downstream oligonucleotide primer introduceda Not 1 site downstream of the termination codon. The general methodused to isolate the soluble AHVSema is described in Spriggs et al., J.Virology, 70:5557 (1996).

[0116] A restriction fragment containing a mutein human Fc region ofimmunoglobulin as described by Goodwin et al. Cell 73, 447-456, 1993 wasligated into an expression vector (pDC409) containing a murine IL-7signal peptide and a FLAG™ octapeptide as described in U.S. Pat. No.5,011,912. The PCR amplified AHVSema DNA encoding was then ligated intothe expression vector containing the mutein human Fc region, the murineIL-7 signal peptide and FLAG™ peptide, in a two way ligation. Theresulting DNA construct was transfected into the monkey kidney celllines CV-1/EBNA (with co-transfection of pSV3neo). After 7 days ofculture in medium containing 0.5% low immunoglobulin bovine serum, asolution of 0.2% azide was added to the supernatant and the supernatantwas filtered through a 0.22 μm filter. Then approximately 1 L of culturesupernatant was passed through a BioCad Protein A HPLC proteinpurification system using a 4.6×100 mm Protein A column (POROS 20A fromPerSeptive Biosystems) at 10 mL/min. The Protein A column binds the FcPortion of the fusion protein in the supernatant, immobilizing thefusion protein and allowing other components of the supernatant to passthrough the column. The column was washed with 30 mL of PBS solution andbound fusion protein was eluted from the HPLC column with citric acidadjusted to pH 3.0. Eluted purified fusions protein was neutralized asit eluted using 1M HEPES solution at pH 7.4.

EXAMPLE 9 Expressing Recombinant Semaphorin Receptor

[0117] Using the semaphorin receptor (VESPR) amino acid sequence of theprotein purified as described in Example 5, and information derived fromEST database searches and cDNAs obtained using hybridizationmethodologies with radiolabeled probes, also as described in Example 5,cDNA is generated and cells are transfected with the cDNA to allowexpression of recombinant VESPR polypeptide.

[0118] The cDNA in DC409 expression vector, derived from pDC406, istransfected in CV1/EBNA cells using standard techniques (McMahan et al.,EMBO J. 10:2821,1991) More particularly, CV1 EBNA cells are plated at adensity of 2×10⁶ cells per 10 cm dish in 10 mL of Dulbeccos MinimumEssential Medium (medium) supplemented with 10% fetal calf serum. Thecells are allowed to adhere overnight at 37° C. The medium is replacedwith 1.5 mL of medium containing 66.7 μM chloroquine and a DNA mixturecontaining 5 μg of cDNA encoding VESPR. Medium containing 175 μL and 25μL of DEAE dextran is added to the cells. The cells and cDNA areincubated at 37° C. for 5 hours. The cDNA mixture is removed and cellsare shocked with 1 mL of fresh medium containing 10% DMSO for 2.5 min.The medium is replaced with fresh medium and the cells are grown for atleast 3 days.

[0119] To recover soluble forms of VESPR, supernatants containing thesoluble form are collected and the VESPR protein recovered using HPLCtechniques or affinity chromatography techniques. To recover forms ofVESPR that are membrane bound, the transfected cells are harvested,fixed in 1% paraformaldehyde, washed and used in their intact form.

EXAMPLE 10 VESPR Binding Studies

[0120] In order to examine the binding characteristics of a receptorpolypeptide of the present invention, binding studies were performed bysubjecting cells expressing membrane bound VESPR extracellular domain tothe slide binding assay described in Goodwin et al. Cell 73:447-456,(1993) and Spriggs et al., J Virol 70:5557 (1996).

[0121] The pDC409 expression vector, derived from pDC406 (McMahon etal., EMBO J. 10:2821, 1991) but having a single Bgl 2 was selected forthe cloning process. VESPR cDNA, encoding amino acids 19-1100, wassubcloned into a pDC409 expression vector through the Sal 1 (5′) and Not1 (3′) sites, to form a DNA construct.

[0122] CV-1/EBNA cells were transfected via DEAE/Dextran with 2 μg of aVESPR cDNA (encoding amino acids 19-1100) in pDC409 (Giri et al., EMBO J13:2822, 1994). The transfected cells were cultured for 3 days and theCV-1/EBNA cell monolayers were incubated with 1 μg/mL of A39R/Fc,AHVSema/Fc, or control Fc protein. Then the incubated cells were washedand incubated with ¹²⁵I-labeled mouse anti-human IgG (JacksonImmunoresearch, West Grove, Pa.). After extensive washing, the cellswere fixed, dipped in photographic emulsion as described by Gearing etal., EMBO J 8:3667-3676 (1989) and developed. Positive binding wasdetermined by the presence of exposed or darkened silver grainsoverlaying cells expressing VESPR that had bound Fc protein.

EXAMPLE 11 Flow Cytometry and Inhibition Binding Studies

[0123] The following describes flow cytometric analyses of CB23 cellsfor binding to A39R/Fc fusion protein (Example 1) and the AHVsema/Fcfusion protein (Example 9). Also described below is a study directed todetermining inhibition of the AHVsema and A39R binding with and anexcess of A39R/polyHis fusion protein prepared as described in Example2.

[0124] The flow cytometric analysis was performed by first incubatingabout 1×10⁶ CB23 cells on ice for 30 minutes in FACS buffer andcontaining 3% normal goat serum and 3% normal rabbit serum to blocknon-specific binding. Portions of A39R/Fc, AHVsema/Fc and a control Fcprotein were added at varying concentrations and the incubation wascontinued for 30 minutes. The cells were washed and then incubated withphycoerythrin-conjugated Fc specific anti-human IgG in FACS buffer. Thecells were washed and analyzed on a FACScan from Becton Dickinson,Bedford, Mass. The results showed positive binding of AHV semaphorin andthe A39R semaphorin.

[0125] Binding inhibition studies were performed by incubating about1×10⁶ CB23 cells for 30 minutes on ice in FACS buffer. The A39R/polyHisand control HIS protein were added to different samples at varyingconcentrations and the incubation continued for another 30 minutes. ThenA39R/Fc or AHVsema/Fc were added to the incubated cells at varyingconcentrations and the incubation was continued for another 30 minutes.The cells were washed and then incubated with phycoerythrin-conjugatedFc specific anti-human IgG in FACS buffers. The cells were washed againand then analyzed on a FACScan. The results demonstrated completeinhibition of A39R and AHVSema using A39R/polyHIS, but not theheterologous HIS containing protein.

EXAMPLE 12 Human B Cell Aggregation with A39R Semaphorin

[0126] In order to examine human B cell response to A39R semaphorin,human tonsillar B cells were purified as described in Spriggs et al., JExp Med 176:1543, (1992). An A39R/polyHis fusion protein was prepared asdescribed in Example 2. A solution of A39R/polyHis fusion protein wasprepared to a final A39R concentration of 1 μg/mL and the A39R/polyHisfusion protein solution was incubated in in vitro cultures of about 10⁵of the purified B cells. Continuing the incubation for about 24 hoursresulted in cellular aggregation. When a 10 fold molar excess of themonoclonal antibody against A39R, prepared as described in Example 6,was added to the fusion protein preparation prior to adding the fusionprotein to the cultures, the cell aggregation was blocked. Additionally,when the A39R semaphorin was heat inactivated prior to adding it to theculture, the aggregation was blocked.

[0127] This work confirms that VESPR is expressed on B cells and thatthe interaction between A39R and VESPR results in B cell aggregation. Bcell aggregation is indicative of their activation. Activated B cellsare known to secrete cytokines, produce antibodies, or become antigenpresenting cells.

EXAMPLE 13 Mouse Dendritic Cells and Macrophage Aggregation with A39RSemaphorin

[0128] In order to examine dendritic cell and macrophage response toA39R, mouse cell cultures were brought into contact with A39R semaphorinand the effects of the combination noted. Mouse dendritic cell culturescontaining macrophages were obtained by immunizing mice with Flt3-L andcells were isolated and purified as described in Maraskovsky et al., JExp Med 184:1953, (1996).

[0129] Briefly, female C57B1/6 mice were injected once daily with asolution of 10 μg of Flt3L and 1 μg mouse serum albumin in 100 μL of PBSfor 9-10 consecutive days. After the immunization, single cellsuspensions of spleens were prepared by disrupting spleen tissue betweenfrosted glass slides in the presence of NH₂Cl to deplete red bloodcells. The remaining cells were incubated with mAb to Thy-1, B220,NK1.1, and TER119, and then incubated with 10% rabbit complement. Thenthe incubated cells were washed and residual mAb-coated cells wereremoved using anti-immunoglobulin (Ig)-coated magnetic beads. Theremaining enriched cells were cultured or sorted for the various cellpopulations.

[0130] Cells selected for sorting were stained with anti-CD11c andanti-CD11b and sorted for the C and D/E populations as described inMaraskovsky et al., J Exp Med 184:1953-1962, 1996.

[0131] An A39R/polyHis fusion protein was prepared as described inExample 2. An A39R/polyHis fusion protein solution was incubated in invitro cultures at a final concentration of 1 μg/mL with about 10⁵ of thesorted or depleted mouse cells. Within 4-6 hours the cells began toaggregate. When a 10 fold molar excess of the monoclonal antibodyagainst A39R, prepared as described in Example 6, was added to theA39R/polyHis fusion protein preparation prior to adding the fusionprotein to the mouse cell cultures, the aggregation was blocked.

[0132] This work confirms that VESPR is expressed on dendritic cells andmacrophages, and that the interaction between A39R and VESPR results indendritic cell and macrophage aggregation.

EXAMPLE 14 A39R Semaphorin Upregulates CD69 Activation Antigen

[0133] In order to investigate the effects of A39R semaphorin oncultured dendritic cells, mice were injected each day for 9 days with aFlt3-L preparation. Mouse dendritic cells were harvested and thencultured in medium containing 10% FBS and 20 ng/mL GM-CSF for 5 days.

[0134] On day 5, 1 μg/mL of A39R/polyHis fusion protein was added to theculture. On day 6, the cells were stained with diagnostic antibodies.The results of the diagnostic antibody staining experiments showed thatCD11c⁺, CD11b⁺ cells (dendritic cells) expressed an increased amount ofthe CD69 activation antigen, thus demonstrating that the interaction ofA39R semaphorin and its receptor upregulate CD69 expression.

[0135] When the fusion protein is inactivated with heat, the fusionprotein had no effect on the CD69 antigen. Representative changes inmean fluorescence intensity between unstained and stained cells werefrom approximately 500 channels to 2500 channels. Again, these resultsdemonstrate significant effects of the interactions between A39Rsemaphorin and its membrane bound receptor on the regulation of the CD69activation antigen, a transient and early expressed marker for cellactivation.

EXAMPLE 15 Evaluating the Effect of A39R in the Production of IL-12

[0136] In order to study the role of A39R in the production of IL-12from mouse spleen cells, mice were immunized with flt3-L and dendriticcells were generated, harvested and purified as described in Example 13.

[0137] Approximately 5×10⁵ cells/0.5 mL of purified, unsorted dendriticcells were incubated in modified DMEM media (500 μL at 1×10⁶/mL) in thepresence of one more of the following: 20 ng/mL muGM-CSF (Immunex,Seattle, Wash.), 20 ng/mL γ-IFN (Genzyme, Boston, Mass.), 10 μg/mL SAC(CalBiochem, La Jolla, Calif.). Each cell preparation was treatedadditionally with 1 μg/mL of A39R/polyHis fusion protein alone or incombination with 1 μg/mL or 0.1 μg/mL of muCD40L trimer (Immunex,Seattle, Wash.). Cultures were incubated in humidified 37C, 10%CO₂-in-air for 16-18 h. After incubation, the viability of each group ofcultured cells was determined and supernatants were collected andassayed for muIL12 (P70) using an ELISA assay kit (Genzyme, Boston,Mass.). MuIL12 levels were calculated by reference to a standard curveconstructed with recombinant cytokine.

[0138] ELISA testing demonstrated in particular that A39R interacts withits receptor to synergize with interferon and SAC in the production ofIL-12 from unsorted mouse dendritic cells. This in vivo IL-12 inductionpromotes natural killer cell activation and gamma interferon productionand contributes to upregulating gamma interferon sensitive cytokines.

EXAMPLE 16 Testing Effects of A39R on Regulation of MHC Class II andCD86 on Monocytes

[0139] The following experiment describes downregulation of MHC Class IIand CD86 by the interaction of A39R with its membrane bound receptor.Peripheral blood from healthy donors was diluted 1:1 in low endotoxinPBS at pH 7.4 and room temperature. Then 35 mLs of the diluted blood waslayered over 15 mLs of Isolymph (Gallard and Schlesinger Industries,Inc; Carle Place, N.Y.) and centrifuged at 2200 rpm for 25 minutes atroom temperature. The plasma layers was reserved. The PBMC layer washarvested and washed three times to remove the Isolymph. The washedPBMC's were resuspended in X-Vivo 15 serum free media (BioWhittaker,Walkersville, Md.) and added to T175 flasks. The flasks had beenpreviously coated with 2% Gelatin (Sigma, St. Louis, Mo.) andpre-treated for 30 minutes with the reserved plasma layer. The PBMC'swere allowed to adhere for 90 minutes at 37° C., 5% CO₂ and then rinsedthree times gently with 10 mL washes of low endotoxin PBS. Adheredmonocytes were harvested by incubating the cells in Enzyme FreeDissociation Buffer (Gibco, BRL) and washing the cells multiple times inPBS. Monocytes were centrifuged at 2500 rpm for 5 minutes, counted, andset up in 24 well dishes at 5×10⁵ cells/well in 1 mL. The cultures were95% pure.

[0140] Purified monocytes were cultured for 7-9 days in the presence of20 ng/mL GM-CSF and 100 ng/mL IL-4 in order to allow cells todifferentiate to a more dendritic cell-like phenotype. On day 7-9,cultures were treated with 1 μg/mL A39R/polyHis or a control polyHiscontaining protein, and the next day cells and supernatants wereharvested for analyses.

[0141] In flow cytometric experiments for examining monocyte-deriveddendritic cell surface markers, cells were stained with conjugated mabsdirected against specific proteins. The staining showed that for amajority of the peripheral blood donors tested, A39R treatmentdownregulated CD86 and MHC class II expression on these cells. SinceCD86 and MHC class II molecules are markers of an enhanced antigenpresentation by dendritic cells, their downregulation suggests animmunosuppressive effect of the interaction of A39R with its receptor onthis cell population.

EXAMPLE 17 Upregulation of CD54

[0142] The following describes the effect of the interaction betweenA39R semaphorin and its receptor on purified monocytes and moreparticularly, the impact of CD54 expression on monocytes afterincubation with a semaphorin. Freshly isolated monocytes were purifiedfrom peripheral blood donors as described in Example 16, except thatthey were held in culture overnight in the presence of A39R/polyHis orcontrol proteins.

[0143] Following the overnight culture, flow cytometry was performedusing the cultured cells and mAbs directed against monocyte specificcell surface markers. In all donors tested, the level of CD54 surfaceexpression was enhanced in the presence of A39R, but not in the presenceof heat inactivated A39R. Similarly, in cultures containing controlproteins CD54 surface expression was not enhanced.

[0144] CD54, also known as ICAM-1, is an adhesion molecule whoseincreased expression is considered to be indicative of cellularactivation. These data indicate that promoting the interaction of A39Rwith its receptor can activate freshly isolated human monocytes.

EXAMPLE 18 Cytokine Induction from Freshly Isolated Human Monocytes

[0145] Freshly isolated human monocytes were purified as described inExample 16, and cultured as described in Example 17. After the overnightincubation with A39R/polyHis, monocyte supernatants were examined forthe presence of proinflammatory cytokines. In all donors tested, IL-6and IL-8 was induced by A39R protein. Heat inactivated A39R and controlproteins did not inducted IL-6 or IL-8. Additionally, cytokineproduction was blocked by the inclusion of a mAb directed against A39R.

[0146] The results of this experiment demonstrate that A39R, orhomologues of this protein, interacting with its receptor, can inducecytokine production by freshly isolated monocytes. Advantageously,soluble forms of VESPR can be used in inhibit the proinflammatoryactivity of monocytes in response to A39R or its homologues.

EXAMPLE 19 Monocyte Aggregation Studies

[0147] In order to examine human monocyte response to the interaction ofa semaphorin to its receptor on monocytes, monocytes were purified asdescribed in Example 17 and an A39R/polyHIS fusion protein was preparedas described in Example 2. The fusion protein and purified, culturedmonocytes were incubated. Continuing the incubation for 20 hoursresulted in monocyte aggregation. In view of the results demonstrated inExample 17, it is suggested that the observed monocyte aggregationoccurs as a result of CD54 upregulation. However, other factors maycontribute to the aggregation as well.

[0148] This work confirms that the semaphorin receptor of the presentinvention is expressed on monocytes and that the interaction betweenA39R and VESPR results in monocyte aggregation. Similar to B cells,monocytes aggregation is indicative of their activation.

EXAMPLE 20 Monoclonal Antibodies to VESPR

[0149] This example illustrates a method for preparing antibodies toVESPR polypeptides. Purified VESPR polypeptide is prepared as describedin Example 10. The purified protein is used to generate antibodiesagainst VESPR as described in U.S. Pat. No. 4,411,993. Briefly, mice areimmunized at 0, 2 and 6 weeks with 10 μg with VESPR. The primaryimmunization is prepared with TITERMAX adjuvant, from Vaxcell, Inc., andsubsequent immunizations are prepared with incomplete Freund's adjuvant(IFA). At 11 weeks, the mice are IV boosted with 3-4 μg VESPR in PBS.Three days after the IV boost, splenocytes are harvested and fused withan Ag8.653 myeloma fusion partner using 50% aqueous PEG 1500 solution.Hybridoma supernatants are screened for VESPR antibodies by dot blotassay against VESPR and an irrelevant Fc protein.

1 10 4707 base pairs nucleic acid double linear cDNA NO NO CDS 1..4707 1ATG GAG GTC TCC CGG AGG AAG GCG CCG CCG CGC CCC CCG CGC CCC GCA 48 MetGlu Val Ser Arg Arg Lys Ala Pro Pro Arg Pro Pro Arg Pro Ala 1 5 10 15GCG CCA CTG CCC CTG CTC GCC TAT CTG CTG GCA CTG GCG GCT CCC GGC 96 AlaPro Leu Pro Leu Leu Ala Tyr Leu Leu Ala Leu Ala Ala Pro Gly 20 25 30 CGGGGC GCG GAC GAG CCC GTG TGG CGG TCG GAG CAA GCC ATC GGA GCC 144 Arg GlyAla Asp Glu Pro Val Trp Arg Ser Glu Gln Ala Ile Gly Ala 35 40 45 ATC GCGGCG AGC CAG GAG GAC GGC GTG TTT GTG GCG AGC GGC AGC TGC 192 Ile Ala AlaSer Gln Glu Asp Gly Val Phe Val Ala Ser Gly Ser Cys 50 55 60 CTG GAC CAGCTG GAC TAC AGC CTG GAG CAC AGC CTC TCG CGC CTG TAC 240 Leu Asp Gln LeuAsp Tyr Ser Leu Glu His Ser Leu Ser Arg Leu Tyr 65 70 75 80 CGG GAC CAAGCG GGC AAC TGC ACA GAG CCG GTC TCG CTG GCG CCC CCC 288 Arg Asp Gln AlaGly Asn Cys Thr Glu Pro Val Ser Leu Ala Pro Pro 85 90 95 GCG CGG CCC CGGCCC GGG AGC AGC TTC AGC AAG CTG CTG CTG CCC TAC 336 Ala Arg Pro Arg ProGly Ser Ser Phe Ser Lys Leu Leu Leu Pro Tyr 100 105 110 CGC GAG GGG GCGGCC GGC CTC GGG GGG CTG CTG CTC ACC GGC TGG ACC 384 Arg Glu Gly Ala AlaGly Leu Gly Gly Leu Leu Leu Thr Gly Trp Thr 115 120 125 TTC GAC CGG GGCGCC TGC GAG GTG CGG CCC CTG GGC AAC CTG AGC CGC 432 Phe Asp Arg Gly AlaCys Glu Val Arg Pro Leu Gly Asn Leu Ser Arg 130 135 140 AAC TCC CTG CGCAAC GGC ACC GAG GTG GTG TCG TGC CAC CCG CAG GGC 480 Asn Ser Leu Arg AsnGly Thr Glu Val Val Ser Cys His Pro Gln Gly 145 150 155 160 TCG ACG GCCGGC GTG GTG TAC CGC GCG GGC CGG AAC AAC CGC TGG TAC 528 Ser Thr Ala GlyVal Val Tyr Arg Ala Gly Arg Asn Asn Arg Trp Tyr 165 170 175 CTG GCG GTGGCC GCC ACC TAC GTG CTG CCT GAG CCG GAG ACG GCG AGC 576 Leu Ala Val AlaAla Thr Tyr Val Leu Pro Glu Pro Glu Thr Ala Ser 180 185 190 CGC TGC AACCCC GCG GCA TCC GAC CAC GAC ACG GCC ATC GCG CTC AAG 624 Arg Cys Asn ProAla Ala Ser Asp His Asp Thr Ala Ile Ala Leu Lys 195 200 205 GAC ACG GAGGGG CGC AGC CTG GCC ACG CAG GAG CTG GGG CGC CTC AAG 672 Asp Thr Glu GlyArg Ser Leu Ala Thr Gln Glu Leu Gly Arg Leu Lys 210 215 220 CTG TGC GAGGGC GCG GGC AGC CTG CAC TTC GTG GAC GCC TTT CTC TGG 720 Leu Cys Glu GlyAla Gly Ser Leu His Phe Val Asp Ala Phe Leu Trp 225 230 235 240 AAC GGCAGC ATC TAC TTC CCC TAC TAC CCC TAC AAC TAT ACG AGC GGC 768 Asn Gly SerIle Tyr Phe Pro Tyr Tyr Pro Tyr Asn Tyr Thr Ser Gly 245 250 255 GCT GCCACC GGC TGG CCC AGC ATG GCG CGC ATC GCG CAG AGC ACC GAG 816 Ala Ala ThrGly Trp Pro Ser Met Ala Arg Ile Ala Gln Ser Thr Glu 260 265 270 GTG CTGTTC CAG GGC CAG GCA TCC CTC GAC TGC GGC CAC GGC CAC CCC 864 Val Leu PheGln Gly Gln Ala Ser Leu Asp Cys Gly His Gly His Pro 275 280 285 GAC GGCCGC CGC CTG CTC CTC TCC TCC AGC CTA GTG GAG GCC CTG GAC 912 Asp Gly ArgArg Leu Leu Leu Ser Ser Ser Leu Val Glu Ala Leu Asp 290 295 300 GTC TGGGCG GGA GTG TTC AGC GCG GCC GCT GGA GAG GGC CAG GAG CGG 960 Val Trp AlaGly Val Phe Ser Ala Ala Ala Gly Glu Gly Gln Glu Arg 305 310 315 320 CGCTCC CCC ACC ACC ACG GCG CTC TGC CTC TTC AGA ATG AGT GAG ATC 1008 Arg SerPro Thr Thr Thr Ala Leu Cys Leu Phe Arg Met Ser Glu Ile 325 330 335 CAGGCG CGC GCC AAG AGG GTC AGC TGG GAC TTC AAG ACG GCC GAG AGC 1056 Gln AlaArg Ala Lys Arg Val Ser Trp Asp Phe Lys Thr Ala Glu Ser 340 345 350 CACTGC AAA GAA GGG GAT CAA CCT GAA AGA GTC CAA CCA ATC GCA TCA 1104 His CysLys Glu Gly Asp Gln Pro Glu Arg Val Gln Pro Ile Ala Ser 355 360 365 TCTACC TTG ATC CAT TCC GAC CTG ACA TCC GTT TAT GGC ACC GTG GTA 1152 Ser ThrLeu Ile His Ser Asp Leu Thr Ser Val Tyr Gly Thr Val Val 370 375 380 ATGAAC AGG ACT GTT TTA TTC TTG GGG ACT GGA GAT GGC CAG TTA CTT 1200 Met AsnArg Thr Val Leu Phe Leu Gly Thr Gly Asp Gly Gln Leu Leu 385 390 395 400AAG GTT ATT CTT GGT GAG AAT TTG ACT TCA AAT TGT CCA GAG GTT ATC 1248 LysVal Ile Leu Gly Glu Asn Leu Thr Ser Asn Cys Pro Glu Val Ile 405 410 415TAT GAA ATT AAA GAA GAG ACA CCT GTT TTC TAC AAA CTC GTT CCT GAT 1296 TyrGlu Ile Lys Glu Glu Thr Pro Val Phe Tyr Lys Leu Val Pro Asp 420 425 430CCT GTG AAG AAT ATC TAC ATT TAT CTA ACA GCT GGG AAA GAG GTG AGG 1344 ProVal Lys Asn Ile Tyr Ile Tyr Leu Thr Ala Gly Lys Glu Val Arg 435 440 445AGA ATT CGT GTT GCA AAC TGC AAT AAA CAT AAA TCC TGT TCG GAG TGT 1392 ArgIle Arg Val Ala Asn Cys Asn Lys His Lys Ser Cys Ser Glu Cys 450 455 460TTA ACA GCC ACA GAC CCT CAC TGC GGT TGG TGC CAT TCG CTA CAA AGG 1440 LeuThr Ala Thr Asp Pro His Cys Gly Trp Cys His Ser Leu Gln Arg 465 470 475480 TGC ACT TTT CAA GGA GAT TGT GTA CAT TCA GAG AAC TTA GAA AAC TGG 1488Cys Thr Phe Gln Gly Asp Cys Val His Ser Glu Asn Leu Glu Asn Trp 485 490495 CTG GAT ATT TCG TCT GGA GCA AAA AAG TGC CCT AAA ATT CAG ATA ATT 1536Leu Asp Ile Ser Ser Gly Ala Lys Lys Cys Pro Lys Ile Gln Ile Ile 500 505510 CGA AGC AGT AAA GAA AAG ACT ACA GTG ACT ATG GTG GGA AGC TTC TCT 1584Arg Ser Ser Lys Glu Lys Thr Thr Val Thr Met Val Gly Ser Phe Ser 515 520525 CCA AGA CAC TCA AAG TGC ATG GTG AAG AAT GTG GAC TCT AGC AGG GAG 1632Pro Arg His Ser Lys Cys Met Val Lys Asn Val Asp Ser Ser Arg Glu 530 535540 CTC TGC CAG AAT AAA AGT CAG CCC AAC CGG ACC TGC ACC TGT AGC ATC 1680Leu Cys Gln Asn Lys Ser Gln Pro Asn Arg Thr Cys Thr Cys Ser Ile 545 550555 560 CCA ACC AGA GCA ACC TAC AAA GAT GTT TCA GTT GTC AAC GTG ATG TTC1728 Pro Thr Arg Ala Thr Tyr Lys Asp Val Ser Val Val Asn Val Met Phe 565570 575 TCC TTC GGT TCT TGG AAT TTA TCA GAC AGA TTC AAC TTT ACC AAC TGC1776 Ser Phe Gly Ser Trp Asn Leu Ser Asp Arg Phe Asn Phe Thr Asn Cys 580585 590 TCA TCA TTA AAA GAA TGC CCA GCA TGC GTA GAA ACT GGC TGC GCG TGG1824 Ser Ser Leu Lys Glu Cys Pro Ala Cys Val Glu Thr Gly Cys Ala Trp 595600 605 TGT AAA AGT GCA AGA AGG TGT ATC CAC CCC TTC ACA GCT TGC GAC CCT1872 Cys Lys Ser Ala Arg Arg Cys Ile His Pro Phe Thr Ala Cys Asp Pro 610615 620 TCT GAT TAT GAG AGA AAC CAG GAA CAG TGT CCA GTG GCT GTC GAG AAG1920 Ser Asp Tyr Glu Arg Asn Gln Glu Gln Cys Pro Val Ala Val Glu Lys 625630 635 640 ACA TCA GGA GGA GGA AGA CCC AAG GAG AAC AAG GGG AAC AGA ACCAAC 1968 Thr Ser Gly Gly Gly Arg Pro Lys Glu Asn Lys Gly Asn Arg Thr Asn645 650 655 CAG GCT TTA CAG GTC TTC TAC ATT AAG TCC ATT GAG CCA CAG AAAGTA 2016 Gln Ala Leu Gln Val Phe Tyr Ile Lys Ser Ile Glu Pro Gln Lys Val660 665 670 TCG ACA TTA GGG AAA AGC AAC GTG ATA GTA ACG GGA GCA AAC TTTACC 2064 Ser Thr Leu Gly Lys Ser Asn Val Ile Val Thr Gly Ala Asn Phe Thr675 680 685 CGG GCA TCG AAC ATC ACA ATG ATC CTG AAA GGA ACC AGT ACC TGTGAT 2112 Arg Ala Ser Asn Ile Thr Met Ile Leu Lys Gly Thr Ser Thr Cys Asp690 695 700 AAG GAT GTG ATA CAG GTT AGC CAT GTG CTA AAT GAC ACC CAC ATGAAA 2160 Lys Asp Val Ile Gln Val Ser His Val Leu Asn Asp Thr His Met Lys705 710 715 720 TTC TCT CTT CCA TCA AGC CGG AAA GAA ATG AAG GAT GTG TGTATC CAG 2208 Phe Ser Leu Pro Ser Ser Arg Lys Glu Met Lys Asp Val Cys IleGln 725 730 735 TTT GAT GGT GGG AAC TGC TCT TCT GTG GGA TCC TTA TCC TACATT GCT 2256 Phe Asp Gly Gly Asn Cys Ser Ser Val Gly Ser Leu Ser Tyr IleAla 740 745 750 CTG CCA CAT TGT TCC CTT ATA TTT CCT GCT ACC ACC TGG ATCAGT GGT 2304 Leu Pro His Cys Ser Leu Ile Phe Pro Ala Thr Thr Trp Ile SerGly 755 760 765 GGT CAA AAT ATA ACC ATG ATG GGC AGA AAT TTT GAT GTA ATTGAC AAC 2352 Gly Gln Asn Ile Thr Met Met Gly Arg Asn Phe Asp Val Ile AspAsn 770 775 780 TTA ATC ATT TCA CAT GAA TTA AAA GGA AAC ATA AAT GTC TCTGAA TAT 2400 Leu Ile Ile Ser His Glu Leu Lys Gly Asn Ile Asn Val Ser GluTyr 785 790 795 800 TGT GTG GCG ACT TAC TGC GGG TTT TTA GCC CCC AGT TTAAAG AGT TCA 2448 Cys Val Ala Thr Tyr Cys Gly Phe Leu Ala Pro Ser Leu LysSer Ser 805 810 815 AAA GTG CGC ACG AAT GTC ACT GTG AAG CTG AGA GTA CAAGAC ACC TAC 2496 Lys Val Arg Thr Asn Val Thr Val Lys Leu Arg Val Gln AspThr Tyr 820 825 830 TTG GAT TGT GGA ACC CTG CAG TAT CGG GAG GAC CCC AGATTC ACG GGG 2544 Leu Asp Cys Gly Thr Leu Gln Tyr Arg Glu Asp Pro Arg PheThr Gly 835 840 845 TAT CGG GTG GAA TCC GAG GTG GAC ACA GAA CTG GAA GTGAAA ATT CAA 2592 Tyr Arg Val Glu Ser Glu Val Asp Thr Glu Leu Glu Val LysIle Gln 850 855 860 AAA GAA AAT GAC AAC TTC AAT ATT TCC AAA AAA GAC ATTGAA ATT ACT 2640 Lys Glu Asn Asp Asn Phe Asn Ile Ser Lys Lys Asp Ile GluIle Thr 865 870 875 880 CTC TTC CAT GGG GAA AAT GGG CAA TTA AAT TGC AGTTTT GAA AAT ATT 2688 Leu Phe His Gly Glu Asn Gly Gln Leu Asn Cys Ser PheGlu Asn Ile 885 890 895 ACT AGA AAT CAA GAT CTT ACC ACC ATC CTT TGC AAAATT AAA GGC ATC 2736 Thr Arg Asn Gln Asp Leu Thr Thr Ile Leu Cys Lys IleLys Gly Ile 900 905 910 AAG ACT GCA AGC ACC ATT GCC AAC TCT TCT AAG AAAGTT CGG GTC AAG 2784 Lys Thr Ala Ser Thr Ile Ala Asn Ser Ser Lys Lys ValArg Val Lys 915 920 925 CTG GGA AAC CTG GAG CTC TAC GTC GAG CAG GAG TCAGTT CCT TCC ACA 2832 Leu Gly Asn Leu Glu Leu Tyr Val Glu Gln Glu Ser ValPro Ser Thr 930 935 940 TGG TAT TTT CTG ATT GTG CTC CCT GTC TTG CTA GTGATT GTC ATT TTT 2880 Trp Tyr Phe Leu Ile Val Leu Pro Val Leu Leu Val IleVal Ile Phe 945 950 955 960 GCG GCC GTG GGG GTG ACC AGG CAC AAA TCG AAGGAG CTG AGT CGC AAA 2928 Ala Ala Val Gly Val Thr Arg His Lys Ser Lys GluLeu Ser Arg Lys 965 970 975 CAG AGT CAA CAA CTA GAA TTG CTG GAA AGC GAGCTC CGG AAA GAG ATA 2976 Gln Ser Gln Gln Leu Glu Leu Leu Glu Ser Glu LeuArg Lys Glu Ile 980 985 990 CGT GAC GGC TTT GCT GAG CTG CAG ATG GAT AAATTG GAT GTG GTT GAT 3024 Arg Asp Gly Phe Ala Glu Leu Gln Met Asp Lys LeuAsp Val Val Asp 995 1000 1005 AGT TTT GGA ACT GTT CCC TTC CTT GAC TACAAA CAT TTT GCT CTG AGA 3072 Ser Phe Gly Thr Val Pro Phe Leu Asp Tyr LysHis Phe Ala Leu Arg 1010 1015 1020 ACT TTC TTC CCT GAG TCA GGT GGC TTCACC CAC ATC TTC ACT GAA GAT 3120 Thr Phe Phe Pro Glu Ser Gly Gly Phe ThrHis Ile Phe Thr Glu Asp 1025 1030 1035 1040 ATG CAT AAC AGA GAC GCC AACGAC AAG AAT GAA AGT CTC ACA GCT TTG 3168 Met His Asn Arg Asp Ala Asn AspLys Asn Glu Ser Leu Thr Ala Leu 1045 1050 1055 GAT GCC CTA ATC TGT AATAAA AGC TTT CTT GTT ACT GTC ATC CAC ACC 3216 Asp Ala Leu Ile Cys Asn LysSer Phe Leu Val Thr Val Ile His Thr 1060 1065 1070 CTT GAA AAG CAG AAGAAC TTT TCT GTG AAG GAC AGG TGT CTG TTT GCC 3264 Leu Glu Lys Gln Lys AsnPhe Ser Val Lys Asp Arg Cys Leu Phe Ala 1075 1080 1085 TCC TTC CTA ACCATT GCA CTG CAA ACC AAG CTG GTC TAC CTG ACC AGC 3312 Ser Phe Leu Thr IleAla Leu Gln Thr Lys Leu Val Tyr Leu Thr Ser 1090 1095 1100 ATC CTA GAGGTG CTG ACC AGG GAC TTG ATG GAA CAG TGT AGT AAC ATG 3360 Ile Leu Glu ValLeu Thr Arg Asp Leu Met Glu Gln Cys Ser Asn Met 1105 1110 1115 1120 CAGCCG AAA CTC ATG CTG AGA CGC ACG GAG TCC GTC GTC GAA AAA CTC 3408 Gln ProLys Leu Met Leu Arg Arg Thr Glu Ser Val Val Glu Lys Leu 1125 1130 1135CTC ACA AAC TGG ATG TCC GTC TGC CTT TCT GGA TTT CTC CGG GAG ACT 3456 LeuThr Asn Trp Met Ser Val Cys Leu Ser Gly Phe Leu Arg Glu Thr 1140 11451150 GTC GGA GAG CCC TTC TAT TTG CTG GTG ACG ACT CTG AAC CAG AAA ATT3504 Val Gly Glu Pro Phe Tyr Leu Leu Val Thr Thr Leu Asn Gln Lys Ile1155 1160 1165 AAC AAG GGT CCC GTG GAT GTA ATC ACT TGC AAA GCC CTG TACACA CTT 3552 Asn Lys Gly Pro Val Asp Val Ile Thr Cys Lys Ala Leu Tyr ThrLeu 1170 1175 1180 AAT GAA GAC TGG CTG TTG TGG CAG GTT CCG GAA TTC AGTACT GTG GCA 3600 Asn Glu Asp Trp Leu Leu Trp Gln Val Pro Glu Phe Ser ThrVal Ala 1185 1190 1195 1200 TTA AAC GTC GTC TTT GAA AAA ATC CCG GAA AACGAG AGT GCA GAT GTC 3648 Leu Asn Val Val Phe Glu Lys Ile Pro Glu Asn GluSer Ala Asp Val 1205 1210 1215 TGT CGG AAT ATT TCA GTC AAT GTT CTC GACTGT GAC ACC ATT GGC CAA 3696 Cys Arg Asn Ile Ser Val Asn Val Leu Asp CysAsp Thr Ile Gly Gln 1220 1225 1230 GCC AAA GAA AAG ATT TTC CAA GCA TTCTTA AGC AAA AAT GGC TCT CCT 3744 Ala Lys Glu Lys Ile Phe Gln Ala Phe LeuSer Lys Asn Gly Ser Pro 1235 1240 1245 TAT GGA CTT CAG CTT AAT GAA ATTGGT CTT GAG CTT CAA ATG GGC ACA 3792 Tyr Gly Leu Gln Leu Asn Glu Ile GlyLeu Glu Leu Gln Met Gly Thr 1250 1255 1260 CGA CAG AAA GAA CTT CTG GACATC GAC AGT TCC TCC GTG ATT CTT GAA 3840 Arg Gln Lys Glu Leu Leu Asp IleAsp Ser Ser Ser Val Ile Leu Glu 1265 1270 1275 1280 GAT GGA ATC ACC AAGCTA AAC ACC ATT GGC CAC TAT GAG ATA TCA AAT 3888 Asp Gly Ile Thr Lys LeuAsn Thr Ile Gly His Tyr Glu Ile Ser Asn 1285 1290 1295 GGA TCC ACT ATAAAA GTC TTT AAG AAG ATA GCA AAT TTT ACT TCA GAT 3936 Gly Ser Thr Ile LysVal Phe Lys Lys Ile Ala Asn Phe Thr Ser Asp 1300 1305 1310 GTG GAG TACTCG GAT GAC CAC TGC CAT TTG ATT TTA CCA GAT TCG GAA 3984 Val Glu Tyr SerAsp Asp His Cys His Leu Ile Leu Pro Asp Ser Glu 1315 1320 1325 GCA TTCCAA GAT GTG CAA GGA AAG AGA CAT CGA GGG AAG CAC AAG TTC 4032 Ala Phe GlnAsp Val Gln Gly Lys Arg His Arg Gly Lys His Lys Phe 1330 1335 1340 AAAGTA AAA GAA ATG TAT CTG ACA AAG CTG CTG TCG ACC AAG GTG GCA 4080 Lys ValLys Glu Met Tyr Leu Thr Lys Leu Leu Ser Thr Lys Val Ala 1345 1350 13551360 ATT CAT TCT GTG CTT GAA AAA CTT TTT AGA AGC ATT TGG AGT TTA CCC4128 Ile His Ser Val Leu Glu Lys Leu Phe Arg Ser Ile Trp Ser Leu Pro1365 1370 1375 AAC AGC AGA GCT CCA TTT GCT ATA AAA TAC TTT TTT GAC TTTTTG GAC 4176 Asn Ser Arg Ala Pro Phe Ala Ile Lys Tyr Phe Phe Asp Phe LeuAsp 1380 1385 1390 GCC CAG GCT GAA AAC AAA AAA ATC ACA GAT CCT GAC GTCGTA CAT ATT 4224 Ala Gln Ala Glu Asn Lys Lys Ile Thr Asp Pro Asp Val ValHis Ile 1395 1400 1405 TGG AAA ACA AAC AGC CTT CCT CTT CGC TTC TGG GTAAAC ATC CTG AAG 4272 Trp Lys Thr Asn Ser Leu Pro Leu Arg Phe Trp Val AsnIle Leu Lys 1410 1415 1420 AAC CCT CAG TTT GTC TTT GAC ATT AAG AAG ACACCA CAT ATA GAC GGC 4320 Asn Pro Gln Phe Val Phe Asp Ile Lys Lys Thr ProHis Ile Asp Gly 1425 1430 1435 1440 TGT TTG TCA GTG ATT GCC CAG GCA TTCATG GAT GCA TTT TCT CTC ACA 4368 Cys Leu Ser Val Ile Ala Gln Ala Phe MetAsp Ala Phe Ser Leu Thr 1445 1450 1455 GAG CAG CAA CTA GGG AAG GAA GCACCA ACT AAT AAG CTT CTC TAT GCC 4416 Glu Gln Gln Leu Gly Lys Glu Ala ProThr Asn Lys Leu Leu Tyr Ala 1460 1465 1470 AAG GAT ATC CCA ACC TAC AAAGAA GAA GTA AAA TCT TAT TAC AAA GCA 4464 Lys Asp Ile Pro Thr Tyr Lys GluGlu Val Lys Ser Tyr Tyr Lys Ala 1475 1480 1485 ATC AGG GAT TTG CCT CCATTG TCA TCC TCA GAA ATG GAA GAA TTT TTA 4512 Ile Arg Asp Leu Pro Pro LeuSer Ser Ser Glu Met Glu Glu Phe Leu 1490 1495 1500 ACT CAG GAA TCT AAGAAA CAT GAA AAT GAA TTT AAT GAA GAA GTG GCC 4560 Thr Gln Glu Ser Lys LysHis Glu Asn Glu Phe Asn Glu Glu Val Ala 1505 1510 1515 1520 TTG ACA GAAATT TAC AAA TAC ATC GTA AAA TAT TTT GAT GAG ATT CTA 4608 Leu Thr Glu IleTyr Lys Tyr Ile Val Lys Tyr Phe Asp Glu Ile Leu 1525 1530 1535 AAT AAACTA GAA AGA GAA CGA GGG CTG GAA GAA GCT CAG AAA CAA CTC 4656 Asn Lys LeuGlu Arg Glu Arg Gly Leu Glu Glu Ala Gln Lys Gln Leu 1540 1545 1550 TTGCAT GTA AAA GTC TTA TTT GAT GAA AAG AAG AAA TGC AAG TGG ATG 4704 Leu HisVal Lys Val Leu Phe Asp Glu Lys Lys Lys Cys Lys Trp Met 1555 1560 1565TAA 4707 * 1568 amino acids amino acid linear protein 2 Met Glu Val SerArg Arg Lys Ala Pro Pro Arg Pro Pro Arg Pro Ala 1 5 10 15 Ala Pro LeuPro Leu Leu Ala Tyr Leu Leu Ala Leu Ala Ala Pro Gly 20 25 30 Arg Gly AlaAsp Glu Pro Val Trp Arg Ser Glu Gln Ala Ile Gly Ala 35 40 45 Ile Ala AlaSer Gln Glu Asp Gly Val Phe Val Ala Ser Gly Ser Cys 50 55 60 Leu Asp GlnLeu Asp Tyr Ser Leu Glu His Ser Leu Ser Arg Leu Tyr 65 70 75 80 Arg AspGln Ala Gly Asn Cys Thr Glu Pro Val Ser Leu Ala Pro Pro 85 90 95 Ala ArgPro Arg Pro Gly Ser Ser Phe Ser Lys Leu Leu Leu Pro Tyr 100 105 110 ArgGlu Gly Ala Ala Gly Leu Gly Gly Leu Leu Leu Thr Gly Trp Thr 115 120 125Phe Asp Arg Gly Ala Cys Glu Val Arg Pro Leu Gly Asn Leu Ser Arg 130 135140 Asn Ser Leu Arg Asn Gly Thr Glu Val Val Ser Cys His Pro Gln Gly 145150 155 160 Ser Thr Ala Gly Val Val Tyr Arg Ala Gly Arg Asn Asn Arg TrpTyr 165 170 175 Leu Ala Val Ala Ala Thr Tyr Val Leu Pro Glu Pro Glu ThrAla Ser 180 185 190 Arg Cys Asn Pro Ala Ala Ser Asp His Asp Thr Ala IleAla Leu Lys 195 200 205 Asp Thr Glu Gly Arg Ser Leu Ala Thr Gln Glu LeuGly Arg Leu Lys 210 215 220 Leu Cys Glu Gly Ala Gly Ser Leu His Phe ValAsp Ala Phe Leu Trp 225 230 235 240 Asn Gly Ser Ile Tyr Phe Pro Tyr TyrPro Tyr Asn Tyr Thr Ser Gly 245 250 255 Ala Ala Thr Gly Trp Pro Ser MetAla Arg Ile Ala Gln Ser Thr Glu 260 265 270 Val Leu Phe Gln Gly Gln AlaSer Leu Asp Cys Gly His Gly His Pro 275 280 285 Asp Gly Arg Arg Leu LeuLeu Ser Ser Ser Leu Val Glu Ala Leu Asp 290 295 300 Val Trp Ala Gly ValPhe Ser Ala Ala Ala Gly Glu Gly Gln Glu Arg 305 310 315 320 Arg Ser ProThr Thr Thr Ala Leu Cys Leu Phe Arg Met Ser Glu Ile 325 330 335 Gln AlaArg Ala Lys Arg Val Ser Trp Asp Phe Lys Thr Ala Glu Ser 340 345 350 HisCys Lys Glu Gly Asp Gln Pro Glu Arg Val Gln Pro Ile Ala Ser 355 360 365Ser Thr Leu Ile His Ser Asp Leu Thr Ser Val Tyr Gly Thr Val Val 370 375380 Met Asn Arg Thr Val Leu Phe Leu Gly Thr Gly Asp Gly Gln Leu Leu 385390 395 400 Lys Val Ile Leu Gly Glu Asn Leu Thr Ser Asn Cys Pro Glu ValIle 405 410 415 Tyr Glu Ile Lys Glu Glu Thr Pro Val Phe Tyr Lys Leu ValPro Asp 420 425 430 Pro Val Lys Asn Ile Tyr Ile Tyr Leu Thr Ala Gly LysGlu Val Arg 435 440 445 Arg Ile Arg Val Ala Asn Cys Asn Lys His Lys SerCys Ser Glu Cys 450 455 460 Leu Thr Ala Thr Asp Pro His Cys Gly Trp CysHis Ser Leu Gln Arg 465 470 475 480 Cys Thr Phe Gln Gly Asp Cys Val HisSer Glu Asn Leu Glu Asn Trp 485 490 495 Leu Asp Ile Ser Ser Gly Ala LysLys Cys Pro Lys Ile Gln Ile Ile 500 505 510 Arg Ser Ser Lys Glu Lys ThrThr Val Thr Met Val Gly Ser Phe Ser 515 520 525 Pro Arg His Ser Lys CysMet Val Lys Asn Val Asp Ser Ser Arg Glu 530 535 540 Leu Cys Gln Asn LysSer Gln Pro Asn Arg Thr Cys Thr Cys Ser Ile 545 550 555 560 Pro Thr ArgAla Thr Tyr Lys Asp Val Ser Val Val Asn Val Met Phe 565 570 575 Ser PheGly Ser Trp Asn Leu Ser Asp Arg Phe Asn Phe Thr Asn Cys 580 585 590 SerSer Leu Lys Glu Cys Pro Ala Cys Val Glu Thr Gly Cys Ala Trp 595 600 605Cys Lys Ser Ala Arg Arg Cys Ile His Pro Phe Thr Ala Cys Asp Pro 610 615620 Ser Asp Tyr Glu Arg Asn Gln Glu Gln Cys Pro Val Ala Val Glu Lys 625630 635 640 Thr Ser Gly Gly Gly Arg Pro Lys Glu Asn Lys Gly Asn Arg ThrAsn 645 650 655 Gln Ala Leu Gln Val Phe Tyr Ile Lys Ser Ile Glu Pro GlnLys Val 660 665 670 Ser Thr Leu Gly Lys Ser Asn Val Ile Val Thr Gly AlaAsn Phe Thr 675 680 685 Arg Ala Ser Asn Ile Thr Met Ile Leu Lys Gly ThrSer Thr Cys Asp 690 695 700 Lys Asp Val Ile Gln Val Ser His Val Leu AsnAsp Thr His Met Lys 705 710 715 720 Phe Ser Leu Pro Ser Ser Arg Lys GluMet Lys Asp Val Cys Ile Gln 725 730 735 Phe Asp Gly Gly Asn Cys Ser SerVal Gly Ser Leu Ser Tyr Ile Ala 740 745 750 Leu Pro His Cys Ser Leu IlePhe Pro Ala Thr Thr Trp Ile Ser Gly 755 760 765 Gly Gln Asn Ile Thr MetMet Gly Arg Asn Phe Asp Val Ile Asp Asn 770 775 780 Leu Ile Ile Ser HisGlu Leu Lys Gly Asn Ile Asn Val Ser Glu Tyr 785 790 795 800 Cys Val AlaThr Tyr Cys Gly Phe Leu Ala Pro Ser Leu Lys Ser Ser 805 810 815 Lys ValArg Thr Asn Val Thr Val Lys Leu Arg Val Gln Asp Thr Tyr 820 825 830 LeuAsp Cys Gly Thr Leu Gln Tyr Arg Glu Asp Pro Arg Phe Thr Gly 835 840 845Tyr Arg Val Glu Ser Glu Val Asp Thr Glu Leu Glu Val Lys Ile Gln 850 855860 Lys Glu Asn Asp Asn Phe Asn Ile Ser Lys Lys Asp Ile Glu Ile Thr 865870 875 880 Leu Phe His Gly Glu Asn Gly Gln Leu Asn Cys Ser Phe Glu AsnIle 885 890 895 Thr Arg Asn Gln Asp Leu Thr Thr Ile Leu Cys Lys Ile LysGly Ile 900 905 910 Lys Thr Ala Ser Thr Ile Ala Asn Ser Ser Lys Lys ValArg Val Lys 915 920 925 Leu Gly Asn Leu Glu Leu Tyr Val Glu Gln Glu SerVal Pro Ser Thr 930 935 940 Trp Tyr Phe Leu Ile Val Leu Pro Val Leu LeuVal Ile Val Ile Phe 945 950 955 960 Ala Ala Val Gly Val Thr Arg His LysSer Lys Glu Leu Ser Arg Lys 965 970 975 Gln Ser Gln Gln Leu Glu Leu LeuGlu Ser Glu Leu Arg Lys Glu Ile 980 985 990 Arg Asp Gly Phe Ala Glu LeuGln Met Asp Lys Leu Asp Val Val Asp 995 1000 1005 Ser Phe Gly Thr ValPro Phe Leu Asp Tyr Lys His Phe Ala Leu Arg 1010 1015 1020 Thr Phe PhePro Glu Ser Gly Gly Phe Thr His Ile Phe Thr Glu Asp 1025 1030 1035 1040Met His Asn Arg Asp Ala Asn Asp Lys Asn Glu Ser Leu Thr Ala Leu 10451050 1055 Asp Ala Leu Ile Cys Asn Lys Ser Phe Leu Val Thr Val Ile HisThr 1060 1065 1070 Leu Glu Lys Gln Lys Asn Phe Ser Val Lys Asp Arg CysLeu Phe Ala 1075 1080 1085 Ser Phe Leu Thr Ile Ala Leu Gln Thr Lys LeuVal Tyr Leu Thr Ser 1090 1095 1100 Ile Leu Glu Val Leu Thr Arg Asp LeuMet Glu Gln Cys Ser Asn Met 1105 1110 1115 1120 Gln Pro Lys Leu Met LeuArg Arg Thr Glu Ser Val Val Glu Lys Leu 1125 1130 1135 Leu Thr Asn TrpMet Ser Val Cys Leu Ser Gly Phe Leu Arg Glu Thr 1140 1145 1150 Val GlyGlu Pro Phe Tyr Leu Leu Val Thr Thr Leu Asn Gln Lys Ile 1155 1160 1165Asn Lys Gly Pro Val Asp Val Ile Thr Cys Lys Ala Leu Tyr Thr Leu 11701175 1180 Asn Glu Asp Trp Leu Leu Trp Gln Val Pro Glu Phe Ser Thr ValAla 1185 1190 1195 1200 Leu Asn Val Val Phe Glu Lys Ile Pro Glu Asn GluSer Ala Asp Val 1205 1210 1215 Cys Arg Asn Ile Ser Val Asn Val Leu AspCys Asp Thr Ile Gly Gln 1220 1225 1230 Ala Lys Glu Lys Ile Phe Gln AlaPhe Leu Ser Lys Asn Gly Ser Pro 1235 1240 1245 Tyr Gly Leu Gln Leu AsnGlu Ile Gly Leu Glu Leu Gln Met Gly Thr 1250 1255 1260 Arg Gln Lys GluLeu Leu Asp Ile Asp Ser Ser Ser Val Ile Leu Glu 1265 1270 1275 1280 AspGly Ile Thr Lys Leu Asn Thr Ile Gly His Tyr Glu Ile Ser Asn 1285 12901295 Gly Ser Thr Ile Lys Val Phe Lys Lys Ile Ala Asn Phe Thr Ser Asp1300 1305 1310 Val Glu Tyr Ser Asp Asp His Cys His Leu Ile Leu Pro AspSer Glu 1315 1320 1325 Ala Phe Gln Asp Val Gln Gly Lys Arg His Arg GlyLys His Lys Phe 1330 1335 1340 Lys Val Lys Glu Met Tyr Leu Thr Lys LeuLeu Ser Thr Lys Val Ala 1345 1350 1355 1360 Ile His Ser Val Leu Glu LysLeu Phe Arg Ser Ile Trp Ser Leu Pro 1365 1370 1375 Asn Ser Arg Ala ProPhe Ala Ile Lys Tyr Phe Phe Asp Phe Leu Asp 1380 1385 1390 Ala Gln AlaGlu Asn Lys Lys Ile Thr Asp Pro Asp Val Val His Ile 1395 1400 1405 TrpLys Thr Asn Ser Leu Pro Leu Arg Phe Trp Val Asn Ile Leu Lys 1410 14151420 Asn Pro Gln Phe Val Phe Asp Ile Lys Lys Thr Pro His Ile Asp Gly1425 1430 1435 1440 Cys Leu Ser Val Ile Ala Gln Ala Phe Met Asp Ala PheSer Leu Thr 1445 1450 1455 Glu Gln Gln Leu Gly Lys Glu Ala Pro Thr AsnLys Leu Leu Tyr Ala 1460 1465 1470 Lys Asp Ile Pro Thr Tyr Lys Glu GluVal Lys Ser Tyr Tyr Lys Ala 1475 1480 1485 Ile Arg Asp Leu Pro Pro LeuSer Ser Ser Glu Met Glu Glu Phe Leu 1490 1495 1500 Thr Gln Glu Ser LysLys His Glu Asn Glu Phe Asn Glu Glu Val Ala 1505 1510 1515 1520 Leu ThrGlu Ile Tyr Lys Tyr Ile Val Lys Tyr Phe Asp Glu Ile Leu 1525 1530 1535Asn Lys Leu Glu Arg Glu Arg Gly Leu Glu Glu Ala Gln Lys Gln Leu 15401545 1550 Leu His Val Lys Val Leu Phe Asp Glu Lys Lys Lys Cys Lys TrpMet 1555 1560 1565 31 base pairs nucleic acid single linear cDNA 3TGTCACTAGT ATCGAATGGC ATAAGTTTGA A 31 33 base pairs nucleic acid singlelinear cDNA NO NO 4 GACAGCGGCC GCCTATTACA TTTTAAGTAT TTT 33 18 basepairs nucleic acid single linear primer NO NO 5 GCGGGACTCA GAGTCACC 1843 base pairs nucleic acid single linear primer NO NO 6 GGATCCTAATACGACTCACT ATAGGGAGGA AACCACTCCG AAC 43 1983 base pairs nucleic acidsingle linear cDNA NO NO CDS 1..1983 7 ATG TTC CAT GTT TCT TTT AGA TATATC TTT GGA ATT CCT CCA CTG ATC 48 Met Phe His Val Ser Phe Arg Tyr IlePhe Gly Ile Pro Pro Leu Ile 1 5 10 15 CTT GTT CTG CTG CCT GTC ACT AGCTCT GAC TAC AAA GAT GAC GAT GAT 96 Leu Val Leu Leu Pro Val Thr Ser SerAsp Tyr Lys Asp Asp Asp Asp 20 25 30 AAA AGA TCT TGT GAC AAA ACT CAC ACATGC CCA CCG TGC CCA GCA CCT 144 Lys Arg Ser Cys Asp Lys Thr His Thr CysPro Pro Cys Pro Ala Pro 35 40 45 GAA GCC GAG GGC GCG CCG TCA GTC TTC CTCTTC CCC CCA AAA CCC AAG 192 Glu Ala Glu Gly Ala Pro Ser Val Phe Leu PhePro Pro Lys Pro Lys 50 55 60 GAC ACC CTC ATG ATC TCC CGG ACC CCT GAG GTCACA TGC GTG GTG GTG 240 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val ThrCys Val Val Val 65 70 75 80 GAC GTG AGC CAC GAA GAC CCT GAG GTC AAG TTCAAC TGG TAC GTG GAC 288 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe AsnTrp Tyr Val Asp 85 90 95 GGC GTG GAG GTG CAT AAT GCC AAG ACA AAG CCG CGGGAG GAG CAG TAC 336 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg GluGlu Gln Tyr 100 105 110 AAC AGC ACG TAC CGT GTG GTC AGC GTC CTC ACC GTCCTG CAC CAG GAC 384 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val LeuHis Gln Asp 115 120 125 TGG CTG AAT GGC AAG GAG TAC AAG TGC AAG GTC TCCAAC AAA GCC CTC 432 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser AsnLys Ala Leu 130 135 140 CCA GCC CCC ATC GAG AAA ACC ATC TCC AAA GCC AAAGGG CAG CCC CGA 480 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys GlyGln Pro Arg 145 150 155 160 GAA CCA CAG GTG TAC ACC CTG CCC CCA TCC CGGGAG GAG ATG ACC AAG 528 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg GluGlu Met Thr Lys 165 170 175 AAC CAG GTC AGC CTG ACC TGC CTG GTC AAA GGCTTC TAT CCC AGC GAC 576 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly PheTyr Pro Ser Asp 180 185 190 ATC GCC GTG GAG TGG GAG AGC AAT GGG CAG CCGGAG AAC AAC TAC AAG 624 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro GluAsn Asn Tyr Lys 195 200 205 ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCCTTC TTC CTC TAT AGC 672 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser PhePhe Leu Tyr Ser 210 215 220 AAG CTC ACC GTG GAC AAG AGC AGG TGG CAG CAGGGG AAC GTC TTC TCA 720 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln GlyAsn Val Phe Ser 225 230 235 240 TGC TCC GTG ATG CAT GAG GCT CTG CAC AACCAC TAC ACG CAG AAG AGC 768 Cys Ser Val Met His Glu Ala Leu His Asn HisTyr Thr Gln Lys Ser 245 250 255 CTC TCC CTG TCT CCG GGT AAA GGA GGG GGCGGA TCA GGG GGC GGA GGA 816 Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly GlySer Gly Gly Gly Gly 260 265 270 TCT ACT AGT ATC GAA TGG CAT AAG TTT GAAACG AGT GAA GAA ATA ATT 864 Ser Thr Ser Ile Glu Trp His Lys Phe Glu ThrSer Glu Glu Ile Ile 275 280 285 TCT ACT TAC TTA ATA GAT GAT GTA TTA TACACG GGC GTT AAT GGG GCG 912 Ser Thr Tyr Leu Ile Asp Asp Val Leu Tyr ThrGly Val Asn Gly Ala 290 295 300 GTA TAT ACA TTT TCA AAT AAT GAA CTA AACAAA ACT GGT TTA ACT AAT 960 Val Tyr Thr Phe Ser Asn Asn Glu Leu Asn LysThr Gly Leu Thr Asn 305 310 315 320 AAC AAT AAT TAT ATC ACA ACA TCT ATAAAA GTA GAG GAT ACA TTA GTA 1008 Asn Asn Asn Tyr Ile Thr Thr Ser Ile LysVal Glu Asp Thr Leu Val 325 330 335 TGC GGA ACC AAT AAC GGA AAC CCC AAATGT TGG AAA ATA GAC GGT TCC 1056 Cys Gly Thr Asn Asn Gly Asn Pro Lys CysTrp Lys Ile Asp Gly Ser 340 345 350 GAA GAT CCA AAA TAT AGA GGT AGA GGATAT GCT CCT TAT CAA AAT AGT 1104 Glu Asp Pro Lys Tyr Arg Gly Arg Gly TyrAla Pro Tyr Gln Asn Ser 355 360 365 AAA GTG ACG ATA ATC AGT CAT AAC GAATGT GTA CTA TCT GAT ATA AAC 1152 Lys Val Thr Ile Ile Ser His Asn Glu CysVal Leu Ser Asp Ile Asn 370 375 380 ATA TCA AAA GAA GGA ATT AAA AGA TGGAGA AGA TTT GAC GGA CCA TGT 1200 Ile Ser Lys Glu Gly Ile Lys Arg Trp ArgArg Phe Asp Gly Pro Cys 385 390 395 400 GGT TAT GAT TTA TAC ACG GCA GATAAC GTG ATT CCA AAA GAT GGT GTG 1248 Gly Tyr Asp Leu Tyr Thr Ala Asp AsnVal Ile Pro Lys Asp Gly Val 405 410 415 CGT GGA GCA TTC GTT GAT AAA GACGGC ACT TAT GAC AAA GTT TAC ATT 1296 Arg Gly Ala Phe Val Asp Lys Asp GlyThr Tyr Asp Lys Val Tyr Ile 420 425 430 CTT TTC ACT GAT ACT ATC GAC ACAAAG AGA ATT GTT AAA ATT CCG TAT 1344 Leu Phe Thr Asp Thr Ile Asp Thr LysArg Ile Val Lys Ile Pro Tyr 435 440 445 ATA GCA CAA ATG TGC TTA AAT GACGAA GGT GGT CCA TCA TCA TTG TCT 1392 Ile Ala Gln Met Cys Leu Asn Asp GluGly Gly Pro Ser Ser Leu Ser 450 455 460 AGT CAT AGA TGG TCG ACG TTT CTCAAG GTC GAA TTA GAA TGT GAT ATC 1440 Ser His Arg Trp Ser Thr Phe Leu LysVal Glu Leu Glu Cys Asp Ile 465 470 475 480 GAC GGA AGA AGT TAT AGA CAAATT ATT CAT TCT AAA GCT ATA AAA ACA 1488 Asp Gly Arg Ser Tyr Arg Gln IleIle His Ser Lys Ala Ile Lys Thr 485 490 495 GAT AAT GAT ACG ATA CTA TATGTA TTC TTT GAT AGT CCT TAT TCC AAG 1536 Asp Asn Asp Thr Ile Leu Tyr ValPhe Phe Asp Ser Pro Tyr Ser Lys 500 505 510 TCC GCA TTA TGT ACC TAT TCTATG AAT GCC ATT AAA CAC TCT TTT TCT 1584 Ser Ala Leu Cys Thr Tyr Ser MetAsn Ala Ile Lys His Ser Phe Ser 515 520 525 ACG TCA AAA TTG GGA GGA TATACA AAG CAA TTG CCG TCT CCA GCT CCT 1632 Thr Ser Lys Leu Gly Gly Tyr ThrLys Gln Leu Pro Ser Pro Ala Pro 530 535 540 GGT ATA TGT CTA CCA GCT GGAAAA GTT GTT CCA CAT ACC ACG TTT GAC 1680 Gly Ile Cys Leu Pro Ala Gly LysVal Val Pro His Thr Thr Phe Asp 545 550 555 560 ATC ATA GAA CAA TAT AATGAG CTA GAT GAT ATT ATA AAG CCT TTA TCT 1728 Ile Ile Glu Gln Tyr Asn GluLeu Asp Asp Ile Ile Lys Pro Leu Ser 565 570 575 CAA CCT ATC TTC GAA GGACCG TCT GGT GTT AAA TGG TTC GAT ATA AAG 1776 Gln Pro Ile Phe Glu Gly ProSer Gly Val Lys Trp Phe Asp Ile Lys 580 585 590 GAG AAG GAA AAT GAA CATCGG GAA TAT AGA ATA TAC TTC ATA AAA GAA 1824 Glu Lys Glu Asn Glu His ArgGlu Tyr Arg Ile Tyr Phe Ile Lys Glu 595 600 605 AAT ACT ATA TAT TCG TTCGAT ACA AAA TCT AAA CAA ACT CGT AGT GCA 1872 Asn Thr Ile Tyr Ser Phe AspThr Lys Ser Lys Gln Thr Arg Ser Ala 610 615 620 CAA GTT GAT GCG CGA CTATTT TCA GTA ATG GTA ACT TCG AAA CCG TTA 1920 Gln Val Asp Ala Arg Leu PheSer Val Met Val Thr Ser Lys Pro Leu 625 630 635 640 TTT ATA GCA GAT ATAGGG ATA GGA GTA GGA ATA CCA CGA ATG AAA AAA 1968 Phe Ile Ala Asp Ile GlyIle Gly Val Gly Ile Pro Arg Met Lys Lys 645 650 655 ATA CTT AAA ATG TAA1983 Ile Leu Lys Met * 660 660 amino acids amino acid linear protein 8Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Ile Pro Pro Leu Ile 1 5 1015 Leu Val Leu Leu Pro Val Thr Ser Ser Asp Tyr Lys Asp Asp Asp Asp 20 2530 Lys Arg Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 35 4045 Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 50 5560 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 65 7075 80 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 8590 95 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr100 105 110 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His GlnAsp 115 120 125 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn LysAla Leu 130 135 140 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys GlyGln Pro Arg 145 150 155 160 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser ArgGlu Glu Met Thr Lys 165 170 175 Asn Gln Val Ser Leu Thr Cys Leu Val LysGly Phe Tyr Pro Ser Asp 180 185 190 Ile Ala Val Glu Trp Glu Ser Asn GlyGln Pro Glu Asn Asn Tyr Lys 195 200 205 Thr Thr Pro Pro Val Leu Asp SerAsp Gly Ser Phe Phe Leu Tyr Ser 210 215 220 Lys Leu Thr Val Asp Lys SerArg Trp Gln Gln Gly Asn Val Phe Ser 225 230 235 240 Cys Ser Val Met HisGlu Ala Leu His Asn His Tyr Thr Gln Lys Ser 245 250 255 Leu Ser Leu SerPro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly 260 265 270 Ser Thr SerIle Glu Trp His Lys Phe Glu Thr Ser Glu Glu Ile Ile 275 280 285 Ser ThrTyr Leu Ile Asp Asp Val Leu Tyr Thr Gly Val Asn Gly Ala 290 295 300 ValTyr Thr Phe Ser Asn Asn Glu Leu Asn Lys Thr Gly Leu Thr Asn 305 310 315320 Asn Asn Asn Tyr Ile Thr Thr Ser Ile Lys Val Glu Asp Thr Leu Val 325330 335 Cys Gly Thr Asn Asn Gly Asn Pro Lys Cys Trp Lys Ile Asp Gly Ser340 345 350 Glu Asp Pro Lys Tyr Arg Gly Arg Gly Tyr Ala Pro Tyr Gln AsnSer 355 360 365 Lys Val Thr Ile Ile Ser His Asn Glu Cys Val Leu Ser AspIle Asn 370 375 380 Ile Ser Lys Glu Gly Ile Lys Arg Trp Arg Arg Phe AspGly Pro Cys 385 390 395 400 Gly Tyr Asp Leu Tyr Thr Ala Asp Asn Val IlePro Lys Asp Gly Val 405 410 415 Arg Gly Ala Phe Val Asp Lys Asp Gly ThrTyr Asp Lys Val Tyr Ile 420 425 430 Leu Phe Thr Asp Thr Ile Asp Thr LysArg Ile Val Lys Ile Pro Tyr 435 440 445 Ile Ala Gln Met Cys Leu Asn AspGlu Gly Gly Pro Ser Ser Leu Ser 450 455 460 Ser His Arg Trp Ser Thr PheLeu Lys Val Glu Leu Glu Cys Asp Ile 465 470 475 480 Asp Gly Arg Ser TyrArg Gln Ile Ile His Ser Lys Ala Ile Lys Thr 485 490 495 Asp Asn Asp ThrIle Leu Tyr Val Phe Phe Asp Ser Pro Tyr Ser Lys 500 505 510 Ser Ala LeuCys Thr Tyr Ser Met Asn Ala Ile Lys His Ser Phe Ser 515 520 525 Thr SerLys Leu Gly Gly Tyr Thr Lys Gln Leu Pro Ser Pro Ala Pro 530 535 540 GlyIle Cys Leu Pro Ala Gly Lys Val Val Pro His Thr Thr Phe Asp 545 550 555560 Ile Ile Glu Gln Tyr Asn Glu Leu Asp Asp Ile Ile Lys Pro Leu Ser 565570 575 Gln Pro Ile Phe Glu Gly Pro Ser Gly Val Lys Trp Phe Asp Ile Lys580 585 590 Glu Lys Glu Asn Glu His Arg Glu Tyr Arg Ile Tyr Phe Ile LysGlu 595 600 605 Asn Thr Ile Tyr Ser Phe Asp Thr Lys Ser Lys Gln Thr ArgSer Ala 610 615 620 Gln Val Asp Ala Arg Leu Phe Ser Val Met Val Thr SerLys Pro Leu 625 630 635 640 Phe Ile Ala Asp Ile Gly Ile Gly Val Gly IlePro Arg Met Lys Lys 645 650 655 Ile Leu Lys Met 660 33 base pairsnucleic acid single linear primer NO NO 9 ATCGCATCAT CTACCTTCATCCATTCCGAC CTG 33 33 base pairs nucleic acid single linear primer NO NO10 TAAACACTCC GAACAGGATT TATGTTTATT GCA 33

What is claimed is:
 1. A method for antagonizing downregulation of theexpression of MHC Class II molecules by dendritic cells comprisingcontacting the dendritic cells with an effective amount of a solubleVESPR protein comprising a VESPR polypeptide consisting of an amino acidsequence at least 90% identical to SEQ ID NO:2, wherein the VESPRpolypeptide can bind to a semaphorin selected from the group consistingof A39R semaphorin and AHV semaphorin.
 2. The method of claim 1, whereinthe soluble VESPR protein is a VESPR polypeptide fusion comprising asecond polypeptide.
 3. The method of claim 2, wherein the secondpolypeptide comprises an Fc region of an antibody.
 4. The method ofclaim 1, wherein the VESPR polypeptide comprises an amino acid sequenceat least 90% identical to amino acids 35-944 of SEQ ID NO:2.
 5. Themethod of claim 4, wherein the VESPR polypeptide comprises amino acids35-944 of SEQ ID NO:2.
 6. The method of claim 1, wherein the dendriticcells are contacted with the soluble VESPR protein in vivo.
 7. Themethod of claim 1, whereby downregulation of the expression of CD86 bythe dendritic cells is antagonized.
 8. The method of claim 1, wherebyupregulation of the expression of CD69 by the dendritic cells isantagonized.
 9. The method of claim 1, whereby upregulation of theexpression of IL-12 by the dendritic cells is antagonized.
 10. A methodfor antagonizing upregulation of the expression of CD54 by monocytescomprising contacting the monocytes with an effective amount of asoluble VESPR protein comprising a VESPR polypeptide consisting of anamino acid sequence at least 90% identical to SEQ ID NO:2, wherein theVESPR polypeptide can bind to a semaphorin selected from the groupconsisting of A39R semaphorin and AHV semaphorin.
 11. The method ofclaim 10, wherein the soluble VESPR protein is a VESPR polypeptidefusion comprising a second polypeptide.
 12. The method of claim 11,wherein the second polypeptide comprises an Fc region of an antibody.13. The method of claim 10, wherein the VESPR polypeptide comprises anamino acid sequence at least 90% identical to amino acids 35-944 of SEQID NO:2.
 14. The method of claim 13, wherein the VESPR polypeptidecomprises amino acids 35-944 of SEQ ID NO:2.
 15. The method of claim 10,wherein the monocytes are contacted with the soluble VESPR protein invivo.
 16. A method for antagonizing upregulation of the expression ofIL-12 by dendritic cells comprising contacting the dendritic cells withan effective amount of a soluble VESPR protein comprising a VESPRpolypeptide consisting of an amino acid sequence at least 90% identicalto SEQ ID NO:2, wherein the VESPR polypeptide can bind to a semaphorinselected from the group consisting of A39R semaphorin and AHVsemaphorin.
 17. The method of claim 16, wherein the soluble VESPRprotein is a VESPR polypeptide fusion comprising a second polypeptide.18. The method of claim 17, wherein the second polypeptide comprises anFc region of an antibody.
 19. The method of claim 16, wherein the VESPRpolypeptide comprises an amino acid sequence at least 90% identical toamino acids 35-944 of SEQ ID NO:2.
 20. The method of claim 19, whereinthe VESPR polypeptide comprises amino acids 35-944 of SEQ ID NO:2. 21.The method of claim 16, wherein the dendritic cells are contacted withthe soluble VESPR protein in vivo.
 22. The method of claim 16, wherebyupregulation of the expression of CD69 by the dendritic cells isantagonized.
 23. The method of claim 16, whereby downregulation of theexpression of MHC Class II molecules and CD86 by the dendritic cells isantagonized.